Novel human calcium dependent proteases, polynucleotides encoding the same, and uses thereof

ABSTRACT

Novel human polunucleotide and polypeptide sequences are disclosed that can be used in therapeuutic, diagnostic, and pharmacogenomic applications.

[0001] The present application is a continuation-in-part of co-pendingU.S. application Ser. No. 09/653,839, filed on Sep. 1, 2000, whichclaims the benefit of U.S. Provisional Application Serial No.60/152,057, filed Sep. 2, 1999, both of which are herein incorporated byreference in their entirety.

1.0 INTRODUCTION

[0002] The present invention relates to the discovery, identification,and characterization of novel human polynucleotides encoding proteinsthat share sequence similarity with human calcium dependent proteases,specifically calpains. The invention encompasses the describedpolynucleotides, host cell expression systems, the encoded proteins,fusion proteins, polypeptides and peptides, antibodies to the encodedproteins and peptides, and genetically engineered animals that eitherlack or overexpress the disclosed polynucleotides, antagonists andagonists of the proteins, and other compounds that modulate theexpression or activity of the proteins encoded by the disclosedpolynucleotides, which can be used for diagnosis, drug screening,clinical trial monitoring, the treatment of diseases and disorders, suchas a reduced white blood cell count, and cosmetic or nutriceuticalapplications.

2.0 BACKGROUND OF THE INVENTION

[0003] Proteases are enzymes that mediate the proteolytic cleavage ofpolypeptide sequences. In particular, calcium-dependent proteases, suchas calpains, have been found in virtually every vertebrate cell that hasbeen examined for their presence. The calpain system has at least threewell-characterized protein members that are activated in response tochanges in calcium concentration. These proteins include at least twocalpains that are activated at different concentrations of calcium, anda calpastatin that specifically inhibits the two calpains. Varioustissue/species specific cDNAs have been described that are homologous tothe calpains. Given the near ubiquitous expression of calpains, theyhave been implicated in a wide variety of cellular functions including,but not limited to, cell proliferation and differentiation, signaltransduction, processes involving interactions between the cell membraneand cytoskeleton secretion, platelet aggregation, cytokinesis, anddisease. Accordingly, calpains represent a key target for the regulationof a variety of biological pathways.

[0004] Reduced white blood cell count, or neutropenia, is a majorcomplication that occurs during many forms of chemotherapy, particularlythose regimens involving myelosuppressive anti-cancer drugs, and as aresult of certain infectious diseases. Although treatments forneutropenia currently exist in the art, they are not ideal for use inall circumstances, and are actually contraindicated in certain patients.Therefore, new treatments for neutropenia would represent a significantadvance in the art.

3.0 SUMMARY OF THE INVENTION

[0005] The present invention relates to the discovery, identification,and characterization of nucleotides that encode novel human proteins,and the corresponding amino acid sequences of these proteins. The novelhuman proteins (NHPs) described for the first time herein sharestructural similarity with animal calcium-activated proteases, orcalpains. As such, the novel genes represent a new class of proteaseproteins with a range of homologues and orthologs that transcend phylaand a broad range of species.

[0006] The novel human nucleic acid sequences described herein, encodeproteins/open reading frames (ORFs) of 739, 723, 702, and 686 aminoacids in length (see SEQ ID NOS:2, 4, 6, and 8 respectively).

[0007] The invention also encompasses agonists and antagonists of thedescribed NHPs, including small molecules, large molecules, mutant NHPs,or portions thereof, that compete with native NHPs, peptides, andantibodies, as well as nucleotide sequences that can be used to inhibitthe expression of the described NHPs (e.g., antisense and ribozymemolecules, and open reading frame or regulatory sequence replacementconstructs) or to enhance the expression of the described NHPs (e.g.,expression constructs that place the described polynucleotide under thecontrol of a strong promoter system), and transgenic animals thatexpress a NHP sequence, or “knock-outs” (which can be conditional) thatdo not express a functional NHP. Knock-out mice can be produced inseveral ways, one of which involves the use of mouse embryonic stem cell(“ES cell”) lines that contain gene trap mutations in a murine homologof at least one of the described NHPs. When the unique NHP sequencesdescribed in SEQ ID NOS:1-9 are “knocked-out” they provide a method ofidentifying phenotypic expression of the particular gene, as well as amethod of assigning function to previously unknown genes. In addition,animals in which the unique NHP sequences described in SEQ ID NOS:1-9are “knocked-out” provide an unique source in which to elicit antibodiesto homologous and orthologous proteins, which would have been previouslyviewed by the immune system as “self” and therefore would have failed toelicit significant antibody responses.

[0008] To these ends, gene trapped knockout ES cells have been generatedin murine homologs of the described NHPs. Characterization of mice inwhich both copies of a NHP have been disrupted (homozygotes) has allowedthe identification of a novel role for this enzyme, and a model for thestudy of certain disorders. In particular, NHP knockout mice (that arehomozygous for the mutated gene) display, intra alia, increased whiteblood cell counts. This suggests that these mice can be used as modelsfor the study of the treatment of a variety of human conditions,including, but not limited to, neutropenia, as exemplified byneutropenia associated with the administration of myelosuppressiveanti-cancer drugs.

[0009] In addition, the invention includes animals containing at least asingle disrupted NHP allele (e.g., “knock-out” mice) that do not expressnormal levels of a NHP, humanized “knock-in” animals where theendogenous murine NHP gene has been replaced by one or morepolynucleotides encoding at least a first human NHP protein, or animalsharboring one or more NHP transgene (e.g., mice overexpressing a NHP).These animals may either transiently, inducibly, or constitutivelyexpress a NHP.

[0010] Additionally, the unique NHP sequences described in SEQ IDNOS:1-9 are useful for the identification of protein coding sequences,and mapping an unique gene to a particular chromosome. These sequencesidentify biologically verified exon splice junctions, as opposed tosplice junctions that may have been bioinformatically predicted fromgenomic sequence alone. The sequences of the present invention are alsouseful as additional DNA markers for restriction fragment lengthpolymorphism (RFLP) analysis, and in forensic biology, particularlygiven the presence of nucleotide polymorphisms within the describedsequences.

[0011] Further, the present invention also relates to processes foridentifying compounds that modulate, i.e., act as agonists orantagonists of, NHP expression and/or NHP activity that utilize purifiedpreparations of the described NHPs and/or NHP products, or cellsexpressing the same. Such compounds can be used as therapeutic agentsfor the treatment of any of a wide variety of symptoms associated withbiological disorders or imbalances, such as reduced white blood cellcount.

[0012] The present invention also provides novel methods andcompositions that can be used to facilitate drug discovery, drugdevelopment, and/or as treatments of conditions such as reduced whiteblood cell count, and the complications resulting therefrom. The presentinvention is based on the identification and novel functionalcharacterization of the NHPs described herein.

[0013] The invention encompasses diagnostic assays that make use of theNHP polynucleotide sequences, or portions thereof, host cells expressingsuch nucleotides, and the expression products of such nucleotides,nucleotides that encode mammalian versions of the NHPs, including humanNHPs, nucleotides that encode NHP mutants and the corresponding mutantNHP expression products, nucleotides that encode portions of a NHP thatcorrespond to one or more of the NHP functional domains and thepolypeptide products specified by such nucleotide sequences, andnucleotides that encode fusion proteins containing a NHP or one or moreof its domains fused to another polypeptide.

[0014] The present invention also features assays for the identificationof compounds that modulate NHP activity in the body. Such compounds canbe used as agents to affect NHP-mediated processes, for example, astherapeutic agents for the treatment of low white blood cell count. Thepresent invention also contemplates methods of using mammalian NHPprotein(s), and particularly recombinantly expressed human NHPprotein(s), in cell-free and/or cell-based assays for identifyingcompounds (modulators) that bind to and/or antagonize or otherwisemodulate (i.e., increase or decrease) NHP activity. Compounds developedusing such assays are then typically used in in vivo assays to determinethe effect of such compounds on NHP-mediated processes, and to discernor verify the observed phenotypic effects. Such phenotypic effectsinclude, but are not limited to, increased white blood cell count orreduction in one or more of the complications associated with reducedwhite blood cell count. The invention thus additionally contemplatescompounds that bind to and/or activate or inhibit the activity of a NHP,as well as pharmaceutical compositions comprising such compounds, andthe use of such compounds to treat NHP-related disorders.

[0015] In addition to small molecule agonists and antagonists of theNHPs, the invention also contemplates the use of large molecules toeffect the levels or bioavailability of a NHP in vivo, including, butnot limited to, mutant NHP proteins that compete with native NHPs,anti-NHP antibodies, anti-idiotypic antibodies that bind anti-NHPantibodies or NHP binding partners, nucleotide sequences that can beused to inhibit NHP expression (e.g., antisense, ribozyme and/or triplexmolecules, and coding sequence or regulatory sequence replacementconstructs) or to enhance NHP expression (e.g., expression constructsthat place a NHP sequence under the control of a strong promoter orexpression system).

[0016] In addition, the invention encompasses methods and compositionsfor the diagnostic evaluation, typing and prognosis of NHP-mediateddisorders, including, inter alia, low white blood cell count, and forthe identification of subjects having a predisposition to suchconditions.

[0017] For example, in another embodiment of the present invention, NHPnucleic acid molecules of the invention can be used as diagnostichybridization probes or as primers for diagnostic PCR analysis for theidentification of NHP gene mutations, allelic variations, and/orregulatory defects in a NHP gene. NHP sequences may be used inhybridization or amplification assays of biological samples to detectabnormalities involving NHP gene structure, including point mutations,insertions, deletions and/or chromosomal rearrangements. Such diagnosticassays include, but are not limited to, Southern analyses, singlestranded conformational polymorphism analyses (SSCP), restrictionfragment length polymorphisms (RFLP), coding single nucleotidepolymorphisms (cSNP) and PCR analyses. These assays can be combined with“gene chip” technology and used to screen pre-existing genetic databasesof patients suffering from various NHP-mediated disorders. The sequencesof the present invention are also useful as additional DNA markers forforensic biology. The present invention further provides for diagnostickits for practicing such methods.

4.0 DESCRIPTION OF THE SEQUENCE LISTING AND FIGURES

[0018] The Sequence Listing provides the sequences of 4 calpain-likeORFs that encode the described NHP amino acid sequences.

5.0 DETAILED DESCRIPTION OF THE INVENTION

[0019] The NHPs, described for the first time herein, are novel proteinsthat are expressed in, inter alia, human cell lines, and human prostateand testis cells. The described sequences were compiled from genetrapped cDNAs and clones isolated from a human testis cDNA library (EdgeBiosystems, Gaithersburg, Md.).

[0020] The present invention encompasses the nucleotides presented inthe Sequence Listing, host cells expressing such nucleotides, thesexpression products of such nucleotides, and: (a) nucleotides thatencode mammalian homologs of the described polynucleotides, includingthe specifically described NHPs, and the NHP products; (b) nucleotidesthat encode one or more portions of the NHPs that correspond tofunctional domains, and the polypeptide products specified by suchnucleotide sequences, including, but not limited to, the novel regionsof any active domain(s); (c) isolated nucleotides that encode mutantversions, engineered or naturally occurring, of the described NHPs, inwhich all or a part of at least one domain is deleted or altered, andthe polypeptide products specified by such nucleotide sequences,including, but not limited to, soluble proteins and peptides; (d)nucleotides that encode chimeric fusion proteins containing all or aportion of a coding region of a NHP, or one of its domains (e.g., areceptor binding domain, accessory protein/self-association domain,etc.) fused to another peptide or polypeptide; or (e) therapeutic ordiagnostic derivatives of the described polynucleotides, such asoligonucleotides, antisense polynucleotides, ribozymes, dsRNA, or genetherapy constructs comprising a sequence first disclosed in the SequenceListing.

[0021] The present invention also includes murine NHPs, mutated muririeembryonic stem cell clones, and animals derived from these embryonicstem cells. Characterization of mice in which NHP gene function has beendisrupted (knock-outs) indicates that the NHPs play a role in conditionsincluding, but not limited to, reduced white blood cell count, one ormore of the complications arising from reduced white blood cell count,and other disorders, as detailed herein.

[0022] The invention encompasses the use of NHP nucleotides, NHPproteins and peptides, as well as antibodies to NHPs (that can, forexample, act as NHP agonists or antagonists), antagonists (peptides,small organic molecules, fusion proteins, etc.) that inhibit NHPactivity or expression, or agonists that activate NHP activityorincrease its expression, in the identification, diagnosis, prognosis,and/or treatment of NHP-mediated disorders. The diagnosis of a NHPabnormality in a patient, or an abnormality in a NHP regulatory pathway,can also facilitate the development of treatments or therapeuticregimens. In addition, NHP nucleotides and NHP proteins can be used toidentify compounds effective in the treatment of, among other things,NHP-mediated disorders, including, but not limited to, reduced whiteblood cell count. In addition, the present invention encompasses methodsand compositions for the diagnostic evaluation, typing and prognosis ofNHP-mediated disorders including, but not limited to, reduced whiteblood cell count.

[0023] An additional embodiment of the present invention relates tomethods of using NHP polynucleotides and/or NHP gene products (proteins,polypeptides and/or peptides) for the identification of compounds thatmodulate, i.e., act as agonists or antagonists, of NHP gene expressionand/or NHP gene product activity. Such compounds can be used as agentsto manipulate NHP-mediated disorders and, in particular, as therapeuticagents for the treatment of NHP-mediated disorders. Such methods andcompositions are typically capable of modulating the level of NHP geneexpression and/or the level of NHP gene product activity. The basis forthese aspects of the present invention is the novel discovery that theelimination of both NHP alleles results in, among other effects,increased white blood cell counts, as shown herein below.

[0024] The invention described in the subsections below thus encompassesNHP polypeptides or peptides corresponding to one or more of thefunctional domains of a NHP, mutated, truncated or deleted NHPs, NHPfusion proteins (e.g., a NHP or one or more functional domains of a NHPfused to an unrelated protein or peptide, such as albumin or animmunoglobulin constant region, i.e., IgFc), nucleotide sequencesencoding such products, and host cell expression systems that canproduce such NHP products.

[0025] The invention also encompasses antibodies and anti-idiotypicantibodies, or fragments thereof (including Fab and F(ab′)₂ fragments),antagonists and agonists of a NHP, as well as compounds or nucleotideconstructs that inhibit expression of a NHP gene (transcription factorinhibitors, antisense and ribozyme molecules, and/or coding sequence orregulatory sequence replacement constructs), or promote expression oroverexpression of a NHP (e.g., expression constructs in which NHP codingsequences are operatively associated with expression control elements,such as promoters, promoter/enhancers, etc.).

[0026] The NHP proteins, polypeptides or peptides, NHP fusion proteins,NHP nucleotide sequences, antibodies, antagonists and/or agonists can beuseful for the detection of mutant NHPs or inappropriately expressedNHPs, which can be used, for example, to diagnose NHP-mediateddisorders. The NHP proteins or peptides, NHP fusion proteins, NHPnucleotide sequences, host cell expression systems, antibodies,antagonists, agonists and genetically engineered cells and animals canalso be used for screening for drugs (or high throughput screening ofcombinatorial libraries) effective in the treatment of the symptomaticor phenotypic manifestations of perturbing the normal function of a NHPin the body. The use of engineered host cells and/or animals can offeran advantage in that such systems allow not only for the identificationof compounds that bind to the endogenous receptor/ligand of a NHP, butcan also identify compounds that trigger NHP-mediated activities orpathways.

[0027] Where, as in the present instance, some of the described NHPpeptides or polypeptides are thought to be cytoplasmic or nuclearproteins, expression systems can be engineered that produce solublederivatives of a NHP (such as those corresponding to NHP extracellularand/or intracellular domains, or truncated NHP polypeptides lacking oneor more hydrophobic domains) and/or NHP fusion protein products(especially NHP-Ig fusion proteins, i.e., fusions of one or more NHPdomain(s) to an IgFc). These expression products, as well as NHP.antibodies, anti-idiotypic antibodies (including Fab fragments), and NHPantagonists or agonists (including compounds that modulate or act ondownstream targets in a NHP-mediated pathway), can be used to directlytreat diseases or disorders. For instance, the administration of aneffective amount of soluble NHP, or a NHP-IgFc fusion protein or ananti-idiotypic antibody (or its Fab) that mimics a NHP could activate oreffectively antagonize the endogenous NHP or a protein interactivetherewith. Nucleotide constructs encoding such NHP products can be usedto genetically engineer host cells to express such products in vivo;these genetically engineered cells function as “bioreactors” in thebody, delivering a continuous supply of a NHP, a NHP peptide, or a NHPfusion protein to the body. Nucleotide constructs encoding functionalNHPs, mutant NHPs, as well as antisense and ribozyme molecules, can alsobe used in “gene therapy” approaches for the modulation of NHPexpression (and, consequently, modulating white blood cell counts higheror lower). Thus, the invention also encompasses pharmaceuticalformulations and methods for treating biological disorders.

[0028] Various aspects of the invention are described in greater detailin the subsections below.

5.1 The NHP Nucleotide Sequences

[0029] The cDNA sequences (SEQ ID NOS:1, 3, 5, 7, and 9) and thecorresponding deduced amino acid sequences (SEQ ID NOS:2, 4, 6, and 8)of the described NHPs are presented in the Sequence Listing. The NHPgenes were obtained from a human testis cDNA library using probes and/orprimers generated from human gene trapped sequence tags. Expressionanalysis has provided evidence that the described NHPs can be expressed,for example, in human testis, prostate, and gene trapped human cells. Inaddition to human calpain genes, the described NHPS share significantsimilarity to a variety of proteases from mice, pigs, chickens, andrats.

[0030] The described open reading frames can also contain severalpolymorphisms, including: an A to G transition corresponding to, forexample, base 1474 of SEQ ID NOS:1 or 3, which can result in either a Kor an E being present at the corresponding amino acid position of SEQ IDNOS:2 or 4; a C to T transition corresponding to, for example, base 1669of SEQ ID NOS:1 or 3, which can result in a Q or a stop codon thattruncates the ORF at the corresponding amino acid position of SEQ IDNOS:2 or 4; and a T to A transversion corresponding to, for example,base, 1673 of SEQ ID NOS:1 or 3, which can result in a L or a H at thecorresponding amino acid position of SEQ ID NOS:2 or 4.

[0031] As discussed above, the present invention includes the human DNAsequences presented in the Sequence Listing (and vectors comprising thesame), and additionally contemplates any nucleotide sequence encoding acontiguous NHP open reading frame (ORF) that hybridizes to a complementof a DNA sequence presented in the Sequence Listing under highlystringent conditions, e.g., hybridization to filter-bound DNA in 0.5 MNaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., andwashing in 0.1×SSC/0.1% SDS at 68° C. (Ausubel et al., eds., 1989,Current Protocols in Molecular Biology, Vol. I, Green PublishingAssociates, Inc., and John Wiley & Sons, Inc., N.Y., at p. 2.10.3) andencodes a functionally equivalent expression product. Additionallycontemplated are any nucleotide sequences that hybridize to thecomplement of a DNA sequence that encodes and expresses an amino acidsequence presented in the Sequence Listing under moderately stringentconditions, e.g., washing in 0.2×SSC/0.1% SDS at 42° C. (Ausubel et al.,1989, supra), yet still encode a functionally equivalent NHP product.Functional equivalents of a NHP include, but are not limited to,naturally occurring NHPs present in other species, and mutant NHPs,whether naturally occurring or engineered (by site directed mutagenesis,gene shuffling, directed evolution as described in, for example, U.S.Pat. No. 5,837,458). The invention also includes degenerate nucleic acidvariants of the disclosed NHP polynucleotide sequences.

[0032] Additionally contemplated are polynucleotides encoding NHP ORFs,or their functional equivalents, encoded by polynucleotide sequencesthat are about 99, 95, 90, or about 85 percent similar to correspondingregions of, for example, SEQ ID NO:1 (as measured by BLAST sequencecomparison analysis using, for example, the University of Wisconsin GCGsequence analysis package (SEQUENCHER 3.0, Gene Codes Corp., Ann Arbor,Mich.) using default parameters).

[0033] The invention also includes nucleic acid molecules, preferablyDNA molecules, that hybridize to, and are therefore the complements of,the described NHP-encoding polynucleotides. Such hybridizationconditions can be highly stringent or less highly stringent, asdescribed herein. In instances where the nucleic acid molecules aredeoxyoligonucleotides (“DNA oligos”), such molecules are generally about16 to about 100 bases long, or about 20 to about 80 bases long, or about34 to about 45 bases long, or any variation or combination of sizesrepresented therein that incorporate a contiguous region of sequencefirst disclosed in the Sequence Listing.

[0034] Although the presently described sequences have been specificallydescribed using nucleotide sequence, it should be appreciated that eachof the sequences can uniquely be described using any of a wide varietyof additional structural attributes, or combinations thereof. Forexample, a given sequence can be described by the net composition of thenucleotides present within a given region of the sequence in conjunctionwith the presence of one or more specific NHP oligonucleotidesequence(s) first disclosed in SEQ ID NOS:1-9. Alternatively, arestriction map specifying the relative positions of restrictionendonuclease digestion sites, or various palindromic or other specificoligonucleotide sequences, can be used to structurally describe a givensequence. Such restriction maps, which are typically generated by widelyavailable computer programs (e.g., the University of Wisconsin GCGsequence analysis package, etc.), can optionally be used in conjunctionwith one or more discrete nucleotide sequence(s) present in the sequencethat can be described by the relative position of the sequence relativeto one or more additional sequence(s), or one or more restriction sites,present in the disclosed sequence.

[0035] These nucleic acid molecules may encode or act as NHP antisensemolecules, useful, for example, in NHP gene regulation and/or asantisense primers in amplification reactions of NHP nucleic acidsequences. With respect to NHP gene regulation, such techniques can beused to regulate one or more of the biological functions associated witha NHP, as described herein. Further, such sequences can be used as partof ribozyme and/or triple helix sequences that are also useful for NHPgene regulation.

[0036] Inhibitory antisense or double stranded oligonucleotides canadditionally comprise at least one modified base moiety that is selectedfrom the group including, but not limited to, 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

[0037] The antisense oligonucleotides can also comprise at least onemodified sugar moiety selected from the group including, but not limitedto, arabinose, 2-fluoroarabinose, xylulose, and hexose.

[0038] In yet another embodiment, the antisense oligonucleotides willcomprise at least one modified phosphate backbone selected from thegroup including, but not limited to a phosphorothioate, aphosphorodithioate, a phosphoramidothioate, a phosphoramidate, aphosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and aformacetal or analog thereof.

[0039] In yet another embodiment, the antisense oligonucleotides areα-anomeric oligonucleotides. An α-anomeric oligonucleotide formsspecific double-stranded hybrids with complementary RNA in which,contrary to the usual β-units, the strands run parallel to each other(Gautier et al., 1987, Nucl. Acids Res. 15:6625-6641). Theoligonucleotide can also be a 2′-0-methylribonucleotide (Inoue et al.,1987, Nucl. Acids Res. 15:6131-6148), or a chimeric RNA-DNA analogue(Inoue et al., 1987, FEBS Lett. 215:327-330). Alternatively, doublestranded RNA can be used to disrupt the expression and function of aNHP.

[0040] Further, NHP homologs and orthologs can be isolated from nucleicacids from additional mammalian species, for example, by performing PCRusing two degenerate or “wobble” oligonucleotide primer pools designedon the basis of amino acid sequences within the NHP sequences disclosedherein. The template for the reaction may be genomic DNA, or total RNA,mRNA, and/or cDNA obtained by reverse transcription of mRNA preparedfrom, for example, human or non-human cell lines, cell types, or tissuesknown to express, or suspected of expressing, an allele of a NHP gene.

[0041] The PCR product can be sequenced directly, or subcloned andsequenced, to ensure that the amplified sequences represent NHP codingsequences. The PCR fragment can then be used to isolate a full lengthcDNA clone by a variety of methods. For example, the amplified fragmentcan be labeled and used to screen a cDNA library, such as abacteriophage cDNA library. Alternatively, the labeled fragment can beused to isolate genomic clones via the screening of a genomic library.

[0042] PCR technology can also be used to isolate full length cDNAsequences. For example, RNA can be isolated, following standardprocedures, from an appropriate cellular or tissue source (i.e., oneknown to express, or suspected of expressing, a NHP). A reversetranscription (RT) reaction can be performed on the RNA using anoligonucleotide primer specific for the most 5′ end of the amplifiedfragment for the priming of first strand synthesis. The resultingRNA/DNA hybrid may then be “tailed” using a standard terminaltransferase reaction, the hybrid may be digested with RNase H, andsecond strand synthesis may then be primed with a complementary primer.Thus, cDNA sequences upstream of the amplified fragment can be isolated.For a review of cloning strategies that can be used, see, e.g., Sambrooket al., 1989, Molecular Cloning, A Laboratory. Manual, Cold SpringHarbor Press, N.Y. (and periodic updates thereof).

[0043] NHP sequences can also be used to isolate mutant alleles of aNHP. Such mutant alleles can be isolated from individuals either knownto have, or suspected of having, a genotype that contributes toincreased white blood cell counts. Mutant alleles and/or peptides,polypeptides or proteins may then be utilized in the therapeutic anddiagnostic programs described herein. Additionally, such sequences ofany of the genes corresponding to NHPs can be used to detect generegulatory (e.g., promoter or promoter/enhancer) defects that canaffect, for example, white blood cell counts.

[0044] A cDNA encoding a mutant NHP gene or sequence can be isolated,for example, by using PCR. In this case, the first cDNA strand may besynthesized by hybridizing an oligo-dT oligonucleotide to mRNA isolatedfrom tissue known to express or suspected of expressing a mutant NHPgene in an individual putatively carrying a mutant NHP allele, and byextending the new strand with reverse transcriptase. The second strandof the cDNA is then synthesized using an oligonucleotide that hybridizesspecifically to the 5′ end of the normal NHP gene. Using these twoprimers, the product is then amplified via PCR, optionally cloned into asuitable vector, and subjected to DNA sequence analysis through methodswell known to those of skill in the art. By comparing the DNA sequenceof the mutant NHP allele to that of a corresponding normal NHP allele,the mutation(s) responsible for the loss or alteration of function ofthe mutant NHP gene product can be ascertained.

[0045] Alternatively, a genomic library can be constructed using DNAobtained from an individual suspected of carrying, or known to carry, amutant NHP allele (e.g., a person manifesting a NHP-associated phenotypesuch as, for example, increased white blood cell counts), or a cDNAlibrary can be constructed using RNA from a tissue known to express, orsuspected of expressing, a mutant NHP allele. A normal NHP gene, or anysuitable fragment thereof, can then be labeled and used as a probe toidentify the corresponding mutant NHP allele in such libraries. Clonescontaining mutant NHP gene sequences can then be purified and subjectedto sequence analysis according to methods well known to those skilled inthe art.

[0046] Additionally, an expression library can be constructed utilizingcDNA synthesized from, for example, RNA isolated from a tissue known toexpress, or suspected of expressing, a mutant NHP allele in anindividual suspected of carrying, or known to carry, such a mutantallele. In this manner, gene products made by the putatively mutanttissue may be expressed and screened using standard antibody screeningtechniques in conjunction with antibodies raised against a normal NHPproduct, as described below (for screening techniques, see, for example,Harlow and Lane, eds., 1988, “Antibodies: A Laboratory Manual”, ColdSpring Harbor Press, Cold Spring Harbor, N.Y., incorporated herein byreference in its entirety).

[0047] Additionally, screening can be accomplished by screening withlabeled NHP fusion proteins, such as, for example, alkalinephosphatase-NHP or NHP-alkaline phosphatase fusion proteins. In caseswhere a NHP mutation results in an expressed gene product with alteredfunction (e.g., as a result of a missense or a frameshift mutation),polyclonal antibodies to a NHP are likely to cross-react with acorresponding mutant NHP gene product. Library clones detected via theirreaction with such labeled antibodies can be purified and subjected tosequence analysis according to methods well known in the art.

[0048] The invention also encompasses nucleotide sequences that encodemutant isoforms of any of the NHP amino acid sequences, peptidefragments thereof, truncated versions thereof, and/or fusion proteins,including any of the above fused to another unrelated polypeptide.Examples of such polypeptides can include, but are not limited to, anepitope tag that aids in purification or detection of the resultingfusion protein, or an enzyme, fluorescent protein, or luminescentprotein that can be used as a marker.

[0049] The present invention additionally encompasses: (a) RNA or DNAvectors that contain any portion of a NHP and/or its complement, as wellas any of the peptides or proteins encoded thereby; (b) DNA vectors thatcontain a cDNA that substantially spans the entire open reading framecorresponding to any of the NHP sequences and/or their complements; (c)DNA expression vectors that contain any of the foregoing sequences, or aportion thereof, operatively associated with a regulatory element thatdirects the expression of the coding sequences in the host cell; and (d)genetically engineered host cells that contain a cDNA that spans theentire open reading frame, or any portion thereof, corresponding to anyof the NHP sequences, operatively associated with a regulatory element,which may be exogenously controlled (such as in gene activation), eitherin vivo and/or in vitro, which directs the expression of NHP codingsequences in the host cell.

[0050] As used herein, regulatory elements include, but are not limitedto, inducible and non-inducible promoters, enhancers, operators, andother elements known to those skilled in the art that drive and regulateexpression. Such regulatory elements include, but are not limited to,the baculovirus polyhedron promoter, the cytomegalovirus (hCMV)immediate early gene promoter, regulatable, viral elements,(particularly retroviral LTR promoters), the early or late promoters ofSV40 and adenovirus, the lac system, the trp system, the TAC system, theTRC system, the major operator and promoter regions of phage lambda, thecontrol regions of fd coat protein, the promoter for 3-phosphoglyceratekinase (PGK), the promoters of acid phosphatase, and the promoters ofthe yeast α-mating factors.

[0051] The present invention also encompasses nucleotide constructsencoding NHP products that can be used to genetically engineer hostcells to express such NHP products in vivo. These genetically engineeredcells function as “bioreactors” in the body, delivering a continuoussupply of a NHP, NHP peptides or polypeptides, soluble NHPs, or NHPfusion proteins. Nucleotide constructs encoding functional versions of aNHP, mutant versions of a NHP, as well as antisense and ribozymemolecules, can be used in “gene therapy” approaches for the modulationof NHP expression and/or activity in the treatment of NHP-mediateddisorders. Thus, the invention also encompasses pharmaceuticalformulations and methods for treating NHP-mediated disorders such asreduced white blood cell count.

[0052] An additional application of the described novel humanpolynucleotide sequences is their use in the molecularmutagenesis/evolution of proteins that are at least partially encoded bythe described novel sequences using, for example, polynucleotideshuffling or related methodologies. Such approaches are described inU.S. Pat. Nos. 5,830,721 and 5,837,458, which are herein incorporated byreference in their entirety.

[0053] 5.1.1 Cells that Contain NHP Disrupted Alleles

[0054] Another aspect of the current invention includes cells thatcontain a disrupted NHP gene. There are a variety of techniques that canbe used to disrupt genes in cells, and especially ES cells. Examples ofsuch methods are described in co-pending U.S. patent application Ser.No. 08/728,963, and U.S. Pat. Nos. 5,789,215, 5,487,992, 5,627,059,5,631,153, 6,087,555, 6,136,566, 6,139,833, and 6,207,371, all of whichare herein incorporated by reference in their entirety.

[0055] 5.1.2 Identification of Cells that Express a NHP

[0056] Host cells that contain NHP coding sequence and/or express abiologically active NHP gene product, or fragment thereof, can beidentified by at least four general approaches: (a) DNA-DNA or DNA-RNAhybridization; (b) the presence or absence of “marker” gene functions;(c) assessing the level of NHP transcription as measured by theexpression of NHP mRNA transcripts in the host cell; and (d) detectionof NHP gene product as measured by immunoassay, enzymatic assay,chemical assay, or one or more of the biological activities of NHPs.These identification methods are described in greater detail below.Prior to screening for gene expression, the host cells can first betreated in an effort to increase the level of expression of sequencesencoding NHP polynucleotides, especially in cell lines that produce lowamounts of NHP mRNAs and/or NHP peptides and proteins.

[0057] In approach (a) above, the presence of a NHP coding sequence canbe detected by DNA-DNA or DNA-RNA hybridization using probes comprisingnucleotide sequences that are homologous or complementary to the NHPcoding sequences, as described herein, or portions or derivativesthereof.

[0058] In approach (b), the recombinant expression vector/host systemcan be identified and selected based upon the presence or absence ofcertain “marker” gene functions (e.g., thymidine kinase activity,resistance to antibiotics, resistance to methotrexate, transformationphenotype, occlusion body formation in baculovirus, etc.). For example,if a NHP polynucleotide sequence that encodes a NHP peptide or proteinis inserted within a marker gene sequence of a vector, recombinantscontaining a NHP coding sequence can be identified by the absence ofmarker gene function. Alternatively, a marker gene can be placed intandem with a NHP sequence, under the control of the same or a differentpromoter used to control the expression of the NHP coding sequence.Expression of the marker gene product in response to induction orselection indicates the presence of the NHP coding sequence.

[0059] In approach (c), transcriptional activity of a coding region of aNHP can be assessed by hybridization assays. For example, RNA can beisolated and analyzed by Northern blot using a probe derived from a NHP,or any portion thereof. Alternatively, total nucleic acids of the hostcell may be extracted and assayed for hybridization to such probes.Additionally, RT-PCR (using NHP specific oligos) may be used to detectlow levels of gene expression in a sample, or in RNA isolated from aspectrum of different tissues, or in cDNA libraries derived fromdifferent tissues, to determine which tissues express a given NHP.

[0060] In approach (d), the expression of the peptides and proteins ofthe current invention can be assessed immunologically, for example byWestern blots, immunoassays such as radioimmuno-precipitation,radioimmunoassays, enzyme-linked immunosorbent assays, and the like.This can be achieved by using an antibody, or a binding partner,specific to a NHP peptide or protein. Additionally, expression can beassessed by monitoring one or more of the biological activities of aNHP. The NHPs have, among others activities, activity as a protease, andis therefore involved in protein degradation. Thus assays describedherein, as well as those commonly known to those of skill in the art toexamine proteases, can be used to access NHP biological activity.

[0061] 5.1.3. The Use NHP Polynucleotide Sequences to DiagnoseNHP-mediated Disorders

[0062] The NHP polynucleotide sequences, as described herein, can beused in hybridization based assays to identify and diagnose NHP-mediateddisorders that result from mutant NHP sequences, or to quantify levelsof NHP expression, thus identifying individuals that are at risk fordeveloping NHP-mediated disorders. These assays could be in the form offluorescence or enzyme based in situ hybridization, PCR, or in apreferred embodiment, hybridization probes used to assess geneexpression patterns using a microarray or high-throughput “chip” format.

[0063] The present invention includes assays that utilize, among others,NHP sequences (and vectors comprising the same), a open reading frame(ORF) encoding a naturally occurring protein having NHP activity andthat hybridizes to a complement of a NHP DNA sequence under highlystringent conditions, as described herein, and encodes a functionallyequivalent gene product, as described herein. The present assays alsocontemplate the use of any nucleotide sequences that hybridize to thecomplement of a nucleotide sequence that encodes a NHP under moderatelystringent conditions, as described herein, yet still encodes afunctionally equivalent NHP product, as described herein.

[0064] The invention also includes the use of nucleic acid molecules,preferably DNA molecules, that hybridize to, and are therefore thecomplements of, the described NHP nucleotide sequences. Suchhybridization conditions may be highly stringent or less highlystringent, as described herein. In instances where the nucleic acidmolecules are “DNA oligos”, such molecules are generally about 16 toabout 100 bases long, or about 20 to about 80 bases long, or about 34 toabout 45 bases long, or any variation or combination of sizesrepresented therein that incorporate a contiguous region of NHPsequence. Such oligonucleotides can be used, for example, in conjunctionwith the polymerase chain reaction (PCR) to screen libraries, isolateclones, and prepare cloning and sequencing templates, etc.

[0065] For oligonucleotide probes, highly stringent conditions cantypically refer, e.g., to washing in 6×SSC/0.05% sodium pyrophosphate at37° C. (for 14-base oligos), 48° C. (for 17-base oligos), 55° C. (for20-base oligos), and 60° C. (for 23-base oligos). Oligonucleotides ofthe invention can be synthesized by standard methods known in the art,e.g., by use of an automated DNA synthesizer (such as are commerciallyavailable from Biosearch, Applied Biosystems, etc.). As examples,phosphorothioate oligonucleotides can be synthesized (Stein et al.,1988, Nucl. Acids Res. 16:3209), and methylphosphonate oligonucleotidescan be prepared by use of controlled pore glass polymer supports (Sarinet al., 1988, Proc. Natl. Acad. Sci. USA 85:7448-7451), etc.

[0066] Low stringency conditions are well-known to those of skill in theart, and will vary predictably depending on the specific organisms fromwhich the library and the labeled sequences are derived. For guidanceregarding such conditions, see, for example, Sambrook, et al., 1989,supra, and Ausubel, et al., 1989, supra (and periodic updates of both).

[0067] Alternatively, NHP oligonucleotides and/or amino acids can beused as hybridization probes for screening libraries, or assessing geneexpression patterns (particularly using a microarray or high-throughput“chip” format). Such assays would be applicable to the screening oflarge databases containing, for example, sequences obtained frompatients suspected of having a NHP defect. This methodology wouldtherefore link functional information with large amounts of geneticinformation.

[0068] Additionally, a series of NHP oligonucleotide sequences, or thecomplements thereof, can be used to represent all or a portion of thedescribed NHP sequences. An oligonucleotide or polynucleotide sequencefirst disclosed in at least a portion of one or more of the sequences ofSEQ ID NOS:1-9 can be used as a hybridization probe in conjunction witha solid support matrix/substrate (resins, beads, membranes, plastics,polymers, metal or metallized substrates, crystalline or polycrystallinesubstrates, etc.). Of particular note are spatially addressable arrays(i.e., gene chips, microtiter plates, etc.) of oligonucleotides andpolynucleotides, or corresponding oligopeptides and polypeptides,wherein at least one of the biopolymers present on the spatiallyaddressable array comprises an oligonucleotide or polynucleotidesequence first disclosed in at least one of the sequences of SEQ IDNOS:1-9, or an amino acid sequence encoded thereby. Methods forattaching biopolymers to, or synthesizing biopolymers on, solid supportmatrices, and conducting binding studies thereon, are disclosed in,inter alia, U.S. Pat. Nos. 5,700,637, 5,556,752, 5,744,305, 4,631,211,5,445,934, 5,252,743, 4,713,326, 5,424,186, and 4,689,405, thedisclosures of which are herein incorporated by reference in theirentirety.

[0069] Addressable arrays comprising sequences first disclosed in SEQ IDNOS:1-9 can be used to identify and characterize the temporal and tissuespecific expression of a gene. These addressable arrays incorporateoligonucleotide sequences of sufficient length to confer the requiredspecificity, yet be within the limitations of the production technology.The length of these probes is usually within a range of between about 8to about 2000 nucleotides. Preferably the probes consist of 60nucleotides, and more preferably 25 nucleotides, from the sequencesfirst disclosed in SEQ ID NOS:1-9.

[0070] For example, a series of NHP oligonucleotide sequences, or thecomplements thereof, can be used in chip format to represent all or aportion of the described NHP sequences. The oligonucleotides, typicallybetween about 16 to about 40 (or any whole number within the statedrange) nucleotides in length, can partially overlap each other, and/orthe sequence may be represented using oligonucleotides that do notoverlap. Accordingly, the described polynucleotide sequences shalltypically comprise at least about two or three distinct oligonucleotidesequences of at least about 8 nucleotides in length that are each firstdisclosed in the described Sequence Listing. Such oligonucleotidesequences can begin at any nucleotide present within a sequence in theSequence Listing, and proceed in either a sense (5′-to-3′) orientationvis-a-vis the described sequence or in an antisense (3′-to-5′)orientation.

[0071] Microarray-based analysis allows the discovery of broad patternsof genetic activity, providing new understanding of gene functions, andgenerating novel and unexepected insight into transcriptional processesand biological mechanisms. The use of addressable arrays comprisingsequences first disclosed in SEQ ID NOS:1-9 provides detailedinformation about transcriptional changes involved in a specificpathway, potentially leading to the identification of novel components,or gene functions that manifest themselves as novel phenotypes.

[0072] Probes consisting of sequences first disclosed in SEQ ID NOS:1-9can also be used in the identification, selection, and validation ofnovel molecular targets for drug discovery. The use of these uniquesequences permits the direct confirmation of drug targets, andrecognition of drug dependent changes in gene expression that aremodulated through pathways distinct from the intended target of thedrug. These unique sequences therefore also have utility in defining andmonitoring both drug action and toxicity.

[0073] As an example of utility, the sequences first disclosed in SEQ IDNOS:1-9 can be utilized in microarrays, or other assay formats, toscreen collections of genetic material from patients who have aparticular medical condition. These investigations can also be carriedout using the sequences first disclosed in SEQ ID NOS:1-9 in silico, andby comparing previously collected genetic databases and the disclosedsequences using computer software known to those in the art. Thus thesequences first disclosed in SEQ ID NOS:1-9 can be used to identifymutations associated with a particular disease, and also in diagnosticor prognostic assays.

[0074] In addition to the NHP nucleotide sequences described herein,additional full length NHP cDNA or gene sequences present in the same orsimilar species (such as, for example, additional splice variants,polymorphisms, pseudogenes, etc.), and/or homologs or orthologs of theNHP gene present in other species, can be identified and readilyisolated by standard molecular biological techniques using the NHPsequences presented herein. The identification of homologs of a NHP inrelated species can be useful, for example, in developing alternativeanimal model systems for the purpose of drug discovery.

[0075] Labeled NHP nucleotide probes can also be used to screen agenomic library derived from an organism of interest, again, usingappropriately stringent conditions. In particular, the identificationand characterization of human genomic clones is helpful for identifyingpolymorphisms (including, but not limited to, nucleotide repeats,microsatellite alleles, single nucleotide polymorphisms, or codingsingle nucleotide polymorphisms), determining the genomic structure of agiven locus/allele, and designing diagnostic tests and clinicalprotocols for treating NHP-related disorders in human patients. Forexample, sequences derived from regions adjacent to the intron/exonboundaries of the human gene can be used to design primers for use inamplification assays to detect mutations within the exons, introns,splice sites (e.g., splice acceptor and/or donor sites), etc., that canbe used in prognostics and/or diagnostics.

[0076] For example, the present sequences can be used in restrictionfragment length polymorphism (RFLP) analysis to identify specificindividuals. In this technique, an individuals genomic DNA is digestedwith one or more restriction enzymes, and probed on a Southern blot toyield unique bands for identification (as generally described in U.S.Pat. No. 5,272,057, incorporated herein by reference). In addition, thesequences of the present invention can be used to provide polynucleotidereagents, e.g., PCR primers, targeted to specific loci in the humangenome, which can enhance the reliability of DNA-based forensicidentifications by, for example, providing another “identificationmarker” (i.e., another DNA sequence that is unique to a particularindividual). Actual base sequence information can be used foridentification as an accurate alternative to patterns formed byrestriction enzyme generated fragments.

5.2 NHP Polypeptides

[0077] NHPs, NHP polypeptides, NHP peptide fragments, mutated,truncated, or deleted forms of the NHPs, and/or NHP fusion proteins canbe prepared for a variety of uses. These uses include, but are notlimited to, the generation of antibodies, as reagents in diagnosticassays, for the identification of other cellular gene products relatedto a NHP, and as reagents in assays for screening for compounds that canbe used as pharmaceutical reagents useful in the therapeutic treatmentof mental, biological, or medical disorders and diseases. Given thesimilarity information and expression data, the described NHPs can betargeted (by drugs, oligos, antibodies, etc.) in order to treat disease,or to augment the efficacy of therapeutic agents.

[0078] The Sequence Listing discloses the amino acid sequences encodedby the described NHP polynucleotide sequences. The NHPs have initiatormethionines in DNA sequence contexts consistent with a translationinitiation site. The sequence data presented herein indicate thatalternatively spliced forms of the NHPs exist (which may or may not betissue specific).

[0079] The NHP amino acid sequences of the invention include thenucleotide and amino acid sequences presented in the Sequence Listing,as well as analogues and derivatives thereof. Further, corresponding NHPhomologues from other species are encompassed by the invention. In fact,any NHP protein encoded by the NHP nucleotide sequences described hereinare within the scope of the invention, as are any novel polynucleotidesequences encoding all or any novel portion of an amino acid sequencepresented in the Sequence Listing. The degenerate nature of the geneticcode is well-known, and, accordingly, each amino acid presented in theSequence Listing is generically representative of the well-known nucleicacid “triplet” codon, or in many cases codons, that can encode the aminoacid. As such, as contemplated herein, the amino acid sequencespresented in the Sequence Listing, when taken together with the geneticcode (see, for example, Table 4-1 at page 109 of “Molecular CellBiology”, 1986, J. Darnell et al., eds., Scientific American Books, NewYork, N.Y., herein incorporated by reference), are genericallyrepresentative of all the various permutations and combinations ofnucleic acid sequences that can encode such amino acid sequences.

[0080] The invention also encompasses proteins that are functionallyequivalent to the NHPs encoded by the presently described nucleotidesequences, as judged by any of a number of criteria, including, but notlimited to, the ability to bind and cleave a substrate of a NHP, or theability to effect an identical or complementary downstream signaltransduction pathway, or a change in cellular metabolism (e.g.,proteolytic activity, ion flux, tyrosine phosphorylation, etc.). Suchfunctionally equivalent NHP proteins include, but are not limited to,additions or substitutions of amino acid residues within the amino acidsequence encoded by the NHP nucleotide sequences described herein, butthat result in a silent change, thus producing a functionally equivalentexpression product. Amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues involved.For example, nonpolar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, proline, phenylalanine, tryptophan, andmethionine; polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine; positivelycharged (basic) amino acids include arginine, lysine, and histidine; andnegatively charged (acidic) amino acids include aspartic acid andglutamic acid.

[0081] While random mutations can be made to NHP DNA (using randommutagenesis techniques well-known in the art), and the resulting NHPmutants tested for activity, site-directed mutations of a NHP codingsequence can be engineered (using site-directed mutagenesis techniqueswell-known to those skilled in the art) to generate NHP mutants withincreased or decreased function.

[0082] For example, the novel amino acid sequence of peptides,polypeptides and proteins encoded by a NHP can be aligned with homologsfrom different species. Mutant peptides, polypeptides and proteins canbe engineered so that regions of interspecies identity are maintained,whereas the variable residues are altered, e.g., by deletion orinsertion of an amino acid residue(s) or by substitution of one or moredifferent amino acid residues. For example, alterations in variableresidues may be designed to produce a mutant form of a NHP peptide,polypeptide or protein that is more stable but retains function. Otheralterations may be designed to alter function, such as those designed toenhance binding or enzymatic activity of a NHP product. One of skill inthe art could easily test such mutant or deleted forms of a NHP peptide,polypeptide or protein for the effect of such alterations on functionusing the teachings presented herein.

[0083] Other mutations to the coding sequences described herein can bemade to generate peptides, polypeptides and proteins that are bettersuited for expression, scale up, etc., in the host cells chosen. Forexample, the triplet code for each amino acid can be modified to conformmore closely to the preferential codon usage of the translationalmachinery of the particular host cell, or, for example, to yield amessenger RNA molecule with a longer half-life. Those skilled in the artwould readily know what modifications of the nucleotide sequence wouldbe desirable to conform the nucleotide sequence to preferential codonusage or to make the messenger RNA more stable. Such information wouldbe obtainable, for example, through use of computer programs, throughreview of available research data on codon usage and messenger RNAstability, and through other means known to those of skill in the art.

[0084] Additionally contemplated are oligopeptides that are modeled onan amino acid sequence first described in the Sequence Listing. Such NHPoligopeptides are generally between about 10 to about 100 amino acidslong, or between about 16 to about 80 amino acids long, or between about20 to about 35 amino acids long, or any variation or combination ofsizes represented therein that incorporate a contiguous region ofsequence first disclosed in the Sequence Listing. Such NHP oligopeptidescan be of any length disclosed within the above ranges and can initiateat any amino acid position represented in the Sequence Listing.

[0085] The invention also contemplates “substantially isolated” or“substantially pure” proteins or polypeptides. By a “substantiallyisolated” or “substantially pure” protein or polypeptide is meant aprotein or polypeptide that has been separated from at least some ofthose components that naturally accompany it. Typically, the protein orpolypeptide is substantially isolated or pure when it is at least 60%,by weight, free from the proteins and other naturally-occurring organicmolecules with which it is naturally associated in vivo. Preferably, thepurity of the preparation is at least 75%, more preferably at least 90%,and most preferably at least 99%, by weight. A substantially isolated orpure protein or polypeptide may be obtained, for example, by extractionfrom a natural source, by expression of a recombinant nucleic acidencoding the protein or polypeptide, or by chemically synthesizing theprotein or polypeptide.

[0086] Purity can be measured by any appropriate method, e.g., columnchromatography such as immunoaffinity chromatography using an antibodyspecific for the protein or polypeptide, polyacrylamide gelelectrophoresis, or HPLC analysis. A protein or polypeptide issubstantially free of naturally associated components when it isseparated from at least some of those contaminants that accompany it inits natural state. Thus, a polypeptide that is chemically synthesized orproduced in a cellular system-different from the cell from which itnaturally originates will be, by definition, substantially free from itsnaturally associated components. Accordingly, substantially isolated orpure proteins or polypeptides include eukaryotic proteins synthesized inE. coli, other prokaryotes, or any other organism in which they do notnaturally occur.

[0087] 5.2.1 NHP Fusion Proteins

[0088] Peptides corresponding to one or more portions of a NHP,truncated or deleted NHPs, as well as fusion proteins in which a fulllength NHP, a NHP Peptide or truncated NHP is fused to an unrelatedprotein are also within the scope of the invention, and can be designedon the basis of NHP nucleotide and/or amino acid sequences disclosedherein. Such fusion proteins include, but are not limited to: IgFcfusions, which stabilize NHP proteins or peptides and prolong half-lifein vivo; fusions to any amino acid sequence that allows the fusionprotein to be anchored to the cell membrane; or fusions to an enzyme,fluorescent protein, or luminescent protein that provides a markerfunction.

[0089] Also encompassed by the present invention are fusion proteinsthat direct a NHP to a target organ and/or facilitate transport acrossthe membrane into the cytosol. Conjugation of NHPs to antibodymolecules, or their Fab or F(ab′)₂ fragments, could be used to targetcells bearing a particular epitope. Attaching an appropriate signalsequence to a NHP would also transport a NHP to a desired locationwithin the cell. Alternatively targeting of a NHP or its nucleic acidsequence might be achieved using liposome or lipid complex baseddelivery systems. Such technologies are described in “Liposomes: APractical Approach”, New, R.R.C., ed., Oxford University Press, N.Y.,and in U.S. Pat. Nos. 4,594,595, 5,459,127, 5,948,767 and 6,110,490 andtheir respective disclosures, which are herein incorporated by referencein their entirety. Additionally embodied are novel protein constructsengineered in such a way that they facilitate transport of NHPs to atarget site or desired organ, where they cross the cell membrane and/orthe nucleus where the NHPs can exert their functional activity. Thisgoal may be achieved by coupling of a NHP to a cytokine or other ligandthat provides targeting specificity, and/or to a protein transducingdomain (see generally U.S. Provisional Patent Application Ser. Nos.60/111,701 and 60/056,713, both of which are herein incorporated byreference, for examples of such transducing sequences), to facilitatepassage across cellular membranes, and can optionally be engineered toinclude nuclear localization signals.

[0090] Alternatively, any fusion protein can be readily purified byutilizing an antibody specific for the fusion protein being expressed.Another exemplary system allows for the ready purification ofnon-denatured fusion proteins expressed in human cell lines (Janknechtet al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-8976). In this system,the sequence of interest is subcloned into a vaccinia recombinationplasmid such that the sequence's open reading frame is translationallyfused to an amino-terminal tag consisting of six histidine residues.Extracts from cells infected with recombinant vaccinia virus are loadedonto Ni²⁺.nitriloacetic acid-agarose columns, and histidine-taggedproteins are selectively eluted with imidazole-containing buffers.

[0091] The novel gene products/peptide sequences encoded by NHPs arealso useful as epitope tags for antigenic or other tagging of proteinsand polypeptides that have been engineered to incorporate or comprise atleast a portion of a NHP peptide sequence.

[0092] 5.2.2 NHP Expression Systems

[0093] While NHP polypeptides and peptides can be chemically synthesized(e.g., see Creighton, 1983, Proteins: Structures and MolecularPrinciples, W. H. Freeman & Co., N.Y.), large polypeptides derived fromNHPs, and full length NHPs themselves, may advantageously be produced byrecombinant DNA technology using techniques well-known in the art forexpressing nucleic acids containing NHP gene sequences and/or codingsequences. Such methods can be used to construct expression vectorscontaining NHP nucleotide sequences and appropriate transcriptional andtranslational control signals. These methods include, for example, invitro recombinant DNA techniques, synthetic techniques, and in vivogenetic recombination (see, for example, the techniques described inSambrook et al., 1989, supra, and Ausubel et al., 1989, supra).Alternatively, RNA and/or DNA encoding NHP nucleotide sequences may bechemically synthesized using, for example, synthesizers (see, forexample, the techniques described in “Oligonucleotide Synthesisi”, 1984,Gait, ed., IRL Press, Oxford, which is incorporated by reference hereinin its entirety).

[0094] A variety of host-expression vector systems can be used toexpress the NHP nucleotide sequences of the invention. Where the NHPpeptide or polypeptide is a soluble derivative of, for example, amembrane protein (e.g., NHP peptides derived from an extracellulardomain (ECD) of a NHP, or truncated or deleted NHPs in which atransmembrane (TM) and/or cytoplasmic domain (CD) have been deleted,etc.) the peptide or polypeptide can be recovered from the culture,i.e., from the host cell in cases where the NHP peptide or polypeptideis not secreted, or from the culture media in cases where the NHPpeptide or polypeptide is secreted by the cells. However, suchexpression systems also encompass engineered host cells that express aNHP, or functional equivalent, in situ, i.e., anchored in the cellmembrane. Purification or enrichment of a NHP from such expressionsystems can be accomplished using appropriate detergents and lipidmicelles and methods well-known to those skilled in the art. However,such engineered host cells themselves may be used in situations where itis important not only to retain the structural and functionalcharacteristics of a NHP, but to assess biological activity, e.g., incertain drug screening assays.

[0095] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines that stablyexpress the NHP sequences described herein may be engineered. Ratherthan using expression vectors that contain viral origins of replication,host cells can be transformed with DNA controlled by appropriateexpression control elements (e.g., promoter, enhancer sequences,transcription terminators, polyadenylation sites, etc.), and aselectable marker. Following the introduction of the foreign DNA,engineered cells may be allowed to grow for 1-2 days in an enrichedmedia, and then switched to a selective media. The selectable marker inthe recombinant plasmid confers resistance to the selection and allowscells to stably integrate the plasmid into their chromosomes and grow toform foci, which in turn can be cloned and expanded into cell lines.This method may advantageously be used to engineer cell lines thatexpress the NHP product. Such engineered cell lines may be particularlyuseful in screening and evaluation of compounds that affect theendogenous activity of the NHP product.

[0096] A number of selection systems may be used, including, but notlimited to, the herpes simplex virus thymidine kinase (Wigler et al.,1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase(Szybalska and Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), andadenine phosphoribosyltransferase (Lowy et al., 1980,Cell 22:817) genes,which can be employed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., 1980, Proc. Natl. Acad. Sci. USA 77:3567; O'Hare et al., 1981,Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance tomycophenolic acid (Mulligan and Berg, 1981, Proc. Natl. Acad. Sci. USA78:2072); neo, which confers resistance to the aminoglycoside G-418(Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1); and hygro, whichconfers resistance to hygromycin (Santerre et al., 1984, Gene 30:147).

[0097] The expression systems that may be used for purposes of theinvention include, but are not limited to, microorganisms such asbacteria (e.g., E. coli, B. subtilis) transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorscontaining NHP nucleotide sequences; yeast (e.g., Saccharomyces, Pichia)transformed with recombinant yeast expression vectors containing NHPnucleotide sequences; insect cell systems infected with recombinantvirus expression vectors (e.g., baculovirus) containing NHP nucleotidesequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing NHP nucleotide sequences; or mammalian cellsystems e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expressionconstructs containing NHP nucleotide sequences and promoters derivedfrom the genome of mammalian cells (e.g., metallothionein promoter) orfrom mammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter).

[0098] In bacterial systems, a number of expression vectors may beadvantageously selected depending-upon the use intended for the NHPproduct being expressed. For example, when a large quantity of such aprotein is to be produced for the generation of pharmaceuticalcompositions of or containing a NHP, or for raising antibodies to a NHP,vectors that direct the expression of high levels of fusion proteinproducts that are readily purified may be desirable. Such vectorsinclude, but are not limited to, the E. coli expression vector pUR278(Ruther et al., 1983, EMBO J. 2:1791), in which a NHP coding sequencemay be ligated individually into the vector in-frame with the lacZcoding region so that a fusion protein is produced; pIN vectors (Inouyeand Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke andSchuster, 1989, J. Biol. Chem. 264:5503-5509); and the like. pGEXvectors may also be used to express foreign polypeptides as fusionproteins with glutathione S-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. The pGEX vectors are designed to includethrombin or factor Xa protease cleavage sites so that the cloned targetexpression product can be released from the GST moiety.

[0099] In an exemplary insect system, Autographa californica nuclearpolyhedrosis virus (AcNPV) is used as a vector tophosphoribosyltransferase (Szybalska and Szybalski, 1962, Proc. Natl.Acad. Sci. USA 48:2026), and adenine phosphoribosyltransferase (Lowy etal., 1980, Cell 22:817) genes, which can be employed in tk⁻, hgprt⁻ oraprt⁻ cells, respectively. Also, antimetabolite resistance can be usedas the basis of selection for the following genes: dhfr, which confersresistance to methotrexate (Wigler et al., 1980, Proc. Natl. Acad. Sci.USA 77:3567; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527);gpt, which confers resistance to mycophenolic acid (Mulligan and Berg,1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistanceto the aminoglycoside G-418 (Colbere-Garapin et al., 1981, J. Mol. Biol.150:1); and hygro, which confers resistance to hygromycin (Santerre etal., 1984, Gene 30:147).

[0100] The expression systems that may be used for purposes of theinvention include, but are not limited to, microorganisms such asbacteria (e.g., E. coli, B. subtilis) transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorscontaining NHP nucleotide sequences; yeast (e.g., Saccharomyces, Pichia)transformed with recombinant yeast expression vectors containing NHPnucleotide sequences; insect cell systems infected with recombinantvirus expression vectors (e.g., baculovirus) containing NHP nucleotidesequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing NHP nucleotide sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expressionconstructs containing NHP nucleotide sequences and promoters derivedfrom the genome of mammalian cells (e.g., metallothionein promoter) orfrom mammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter). translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see Bitter et al., 1987,Methods in Enzymol. 153:516-544).

[0101] In yeast, a number of vectors containing constitutive orinducible promoters may be used. For a review, see Ausubel et al., 1989,supra, Ch. 13; Grant et al., 1987, Methods in Enzymol. 153:516-544;Glover, 1986, DNA Cloning, Vol. II, IRL Press, Wash., D.C., Ch. 3;Bitter, 1987, Methods in Enzymol. 152:673-684; and Strathern et al.,eds., “The Molecular Biology of the Yeast Saccharomyces”, 1982, ColdSpring Harbor Press, Vols. I and II.

[0102] In cases where plant expression vectors are used, expression of aNHP coding sequence may be driven by any of a number of promoters. Forexample, viral promoters such as the 35S RNA-and 19S RNA promoters ofCaMV (Brisson et al., 1984, Nature 310:511-514), or the coat proteinpromoter of TMV. (Takamatsu et al., 1987, EMBO J. 6:307-311) may beused; alternatively, plant promoters such as the small subunit ofRUBISCO (Coruzzi et al., 1984, EMBO J. 3:1671-1680; Broglie et al.,1984, Science 224:838-843); or heat shock promoters, e.g., soybeanhsp17.5-E or hsp17.3-B (Gurley et al., 1986, Mol. Cell. Biol. 6:559-565)may be used. These constructs can be introduced into plant cells usingTi plasmids, Ri plasmids, plant virus vectors, direct DNAtransformation, microinjection, electroporation, etc. For reviews ofsuch techniques, see, for example, Weissbach and Weissbach, 1988,Methods for Plant Molecular Biology, Academic Press, NY, Section VIII,pp. 421-463; and Grierson and Corey, 1988, Plant Molecular Biology, 2dEd., Blackie, London, Ch. 7-9.

[0103] In addition, a host cell strain may be chosen that modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correct ordesired modification and processing of the NHP-protein, polypeptide orpeptide expressed. To this end, eukaryotic host cells that possess thecellular machinery for proper processing of the primary transcript,glycosylation, and phosphorylation of the NHP gene product may be used.Such mammalian host cells include, but are not limited to, CHO, VERO,BHK, HeLa, COS, MDCK, 293, 3T3, WI38 and U937 cells, and in particularhuman cell lines.

[0104] 5.2.3 NHP Transgenic Animals

[0105] The present invention provides for transgenic animals that carrya NHP transgene in all their cells, as well as animals that carry a NHPtransgene in some, but not all their cells, i.e., mosaic animals orsomatic cell transgenic animals. Animals of any species, including, butnot limited to, mice, rats, rabbits, guinea pigs, pigs, micro-pigs,goats, and non-human primates, e.g., baboons, monkeys, and chimpanzees,can be used to generate transgenic animals carrying NHP polynucleotides.NHP transgenes may be integrated as a single transgene or inconcatamers, e.g., head-to-head tandems or head-to-tail tandems. Thetransgene may also be selectively introduced into and activated in aparticular cell type by following, for example, the teaching of Lasko etal., 1992, Proc. Natl. Acad. Sci. USA 89:6232-6236. The regulatorysequences required for such a cell-type specific activation will dependupon the particular cell type of interest, and will be apparent to thoseof skill in the art.

[0106] When it is desired that a NHP transgene be integrated into thechromosomal site of the endogenous copy of the NHP gene, gene targetingis preferred. Briefly, when such a technique is to be utilized, vectorscontaining some nucleotide sequences homologous to the endogenous NHPgene are designed for the purpose of integrating, via homologousrecombination with chromosomal sequences, into and disrupting thefunction of the nucleotide sequence of the endogenous NHP gene (i.e.,“knockout” animals). In this way, the expression of the endogenous NHPgene may also be eliminated by inserting nonfunctional sequences intothe endogenous NHP gene. The transgene may also be selectivelyintroduced into a particular cell type, thus inactivating the endogenousNHP gene in only that cell type, by following, for example, the teachingof Gu et al., 1994, Science 265:103-106. The regulatory sequencesrequired for such a cell-type specific inactivation will depend upon theparticular cell type of interest.

[0107] Any technique known in the art may be used to introduce a NHPtransgene into animals to produce the founder lines of transgenicanimals. Such techniques include, but are not limited to, pronuclearmicroinjection (U.S. Pat. No. 4,873,191, incorporated herein byreference); retrovirus-mediated gene transfer into germ lines (Van derPutten et al., 1985, Proc. Natl. Acad. Sci. USA 82:6148-6152); genetargeting in embryonic stem cells.(Thompson et al., 1989, Cell56:313-321); electroporation of embryos (Lo, 1983, Mol Cell. Biol.3:1803-1814); sperm-mediated gene transfer (Lavitrano et al., 1989, Cell57:717-723); and positive-negative selection as described in U.S. Pat.No. 5,464,764, herein incorporated by reference. For a review of suchtechniques, see Gordon, 1989, Transgenic Animals, Intl. Rev. Cytol.115:171-229, which is incorporated by reference herein in its entirety.

[0108] Once transgenic animals have been generated, the expression ofthe recombinant NHP gene may be assayed utilizing standard techniques.Initial screening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to assay whether integration of theNHP transgene has taken place. The level of mRNA expression of the NHPtransgene in the tissues of the transgenic animals may also be assessedusing techniques that include, but are not limited to, Northern blotanalysis of cell type samples lines or host systems can be chosen toensure the correct or desired modification and processing of the NHPprotein, polypeptide or peptide expressed. To this end, eukaryotic hostcells that possess the cellular machinery for proper processing of theprimary transcript, glycosylation, and phosphorylation of the NHP geneproduct may be used. Such mammalian host cells include, but are notlimited to, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38 and U937cells, and in particular human cell lines.

[0109] 5.2.3 NHP Transgenic Animals

[0110] The present invention provides for transgenic animals that carrya NHP transgene in all their cells, as well as animals that carry a NHPtransgene in some, but not all their cells, i.e., mosaic animals orsomatic cell transgenic animals. Animals of any species, including, butnot limited to, mice, rats, rabbits, guinea pigs, pigs, micro-pigs,goats, and non-human primates, e.g., baboons, monkeys, and chimpanzees,can be used to generate transgenic animals carrying NHP polynucleotides.NHP transgenes may be integrated as a single transgene or inconcatamers, e.g., head-to-head tandems or head-to-tail tandems. Thetransgene may also be selectively introduced into and activated in aparticular cell type by following, for example, the teaching of Lakso etal., 1992, Proc. Natl. Acad. Sci. USA 89:6232-6236. The regulatorysequences required for such a cell-type specific activation will dependupon the particular cell type of interest, and will be apparent to thoseof skill in the art.

[0111] When it is desired that a NHP transgene be integrated into thechromosomal site of the endogenous copy of the NHP gene, gene targetingis preferred. Briefly, when such a technique is to be utilized, vectorscontaining some nucleotide sequences homologous to the endogenous NHPgene are designed for the purpose of integrating, via homologousrecombination with chromosomal sequences, into and disrupting thefunction of the nucleotide and/or activity of a NHP expression product.Additionally, such antibodies can be used in conjunction with genetherapy to, for example, evaluate normal and/or engineeredNHP-expressing cells prior to their introduction into a patient. Suchantibodies may additionally be used in methods for the inhibition ofabnormal NHP activity. Thus, such antibodies may be utilized as a partof treatment methods.

[0112] For the production of antibodies, various host animals may beimmunized by injection with a NHP, a NHP peptide (e.g., onecorresponding to a functional domain of a NHP), a truncated NHPpolypeptide (a NHP in which one or more domains have been deleted),functional equivalents of a NHP, or mutated variants of a NHP. Such hostanimals may include, but are not limited to, pigs, rabbits, mice, goats,and rats, to name but a few. Various adjuvants may be used to increasethe immunological response, depending on the host species, including,but not limited to, Freund's adjuvant (complete and incomplete), mineralsalts such as aluminum hydroxide or aluminum phosphate, chitosan,surface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and Corynebacteriumparvum. Alternatively, the immune response could be enhanced bycombination and/or coupling with molecules such as keyhole limpethemocyanin, tetanus toxoid, diphtheria toxoid, ovalbumin, cholera toxin,or fragments thereof. Polyclonal antibodies are heterogeneouspopulations of antibody molecules derived from the sera of the immunizedanimals.

[0113] Monoclonal antibodies, which are homogeneous populations ofantibodies to a particular antigen, may be obtained by any techniquethat provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to, thehybridoma technique of Kohler and Milstein (1975, Nature 256:495-497;and U.S. Pat. No. 4,376,110), the human B-cell hybridoma technique(Kosbor et al., 1983, Immunology Today 4:72; Cole et al., 1983, Proc.Natl. Acad. Sci. USA 80:2026-2030), and the EBV-hybridoma technique(Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy, Alan R.Liss, Inc., pp. 77-96). Such antibodies may be of any immunoglobulinclass, including IgG, IgM, IgE, IgA, and IgD, and any subclass thereof.The hybridomas producing the mAbs of this invention may be cultivated invitro or in vivo. Production of high titers of mAbs in vivo makes thisthe presently preferred method of production.

[0114] In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci. USA81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda etal., 1985, Nature, 314:452-454) by splicing the genes from a mouseantibody molecule of appropriate antigen specificity together with genesfrom a human antibody molecule of appropriate biological activity can beused. A chimeric antibody is a molecule in which different portions arederived from different animal species, such as those having a variableregion derived from a murine mAb and a human immunoglobulin constantregion. Such technologies are described in U.S. Pat. Nos. 6,114,598,6,075,181 and 5,877,397 and their respective disclosures, which areherein incorporated by reference in their entirety. Also encompassed bythe present invention is the use of fully humanized monoclonalantibodies, as described in U.S. Pat. No. 6,150,584 and respectivedisclosures, which are herein incorporated by reference in theirentirety.

[0115] Alternatively, techniques described for the production of singlechain antibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA85:5879-5883; and Ward et al., 1989, Nature 341:544-546) can be adaptedto produce single chain antibodies against NHP expression products.Single chain antibodies are formed by linking the heavy and light chainfragments of the Fv region via an amino acid bridge, resulting in asingle chain polypeptide.

[0116] Antibody fragments that recognize specific epitopes may begenerated by known techniques. For example, such fragments include, butare not limited to: F(ab′)₂ fragments, which can be produced by pepsindigestion of an antibody molecule; and Fab fragments, which can begenerated by reducing the disulfide bridges of F(ab′)₂ fragments.Alternatively, Fab expression libraries may be constructed (Huse et al.,1989, Science, 246:1275-1281) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

[0117] Antibodies to a NHP can, in turn be utilized to generateanti-idiotype antibodies that “mimic” a given NHP, using techniqueswell-known to those skilled in the art (see, e.g., Greenspan and Bona,1993, FASEB J. 7:437-444; and Nissinoff, 1991, J. Immunol.147:2429-2438). For example, antibodies that bind to a NHP domain andcompetitively inhibit the binding of NHP to its cognate receptor can beused to-generate anti-idiotypes that “mimic” the NHP and, therefore,bind and activate or neutralize a receptor. Such anti-idiotypicantibodies or fragments of such anti-idiotypes can be used intherapeutic regimens involving a NHP signaling pathway.

[0118] Additionally given the high degree of relatedness of mammalianNHPs, the presently described knock-out mice (having never seen a NHP,and thus never been tolerized to a NHP) have an unique utility, as theycan be advantageously applied to the generation of antibodies againstthe disclosed mammalian NHPs (i.e., a NHP will be immunogenic in NHPknock-out animals).

5.4 Diagnosis of NHP-mediated Disorders

[0119] A variety of methods can be employed for the diagnostic andprognostic evaluation of NHP-mediated disorders. These methods can alsobe used to identify subjects having a predisposition to such disorders.Such methods may, for example, utilize reagents such as NHP nucleotidesequences, NHP proteins or peptides, and/or anti-NHP antibodies.Specifically, such reagents may be used, for example, for: (1) thedetection of the presence of NHP gene mutations, or the detection ofeither over- or under-expression of a NHP mRNA relative to the non-NHPdisorder state; (2) the detection of either an over- or anunder-abundance of a NHP gene product Nature 256:495-497; and U.S. Pat.No. 4,376,110), the human B-cell hybridoma technique (Kosbor et al.,1983, Immunology Today 4:72; Cote et al., 1983, Proc. Natl. Acad. Sci.USA 80:2026-2030), and the EBV-hybridoma technique (Cole et al., 1985,Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Such antibodies may be of any immunoglobulin class, incudingIgG, IgM, IgE, IgA, and IgD, and any subclass thereof. The hybridomasproducing the mAbs of this invention may be cultivated in vitro or invivo. Production of high titers of mAbs in vivo makes this the presentlypreferred method of production.

[0120] In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci. USA81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda etal., 1985, Nature, 314:452-454) by splicing the genes from a mouseantibody molecule of appropriate antigen specificity together with genesfrom a human antibody molecule of appropriate biological activity can beused. A chimeric antibody is a molecule in which different portions arederived from different animal species, such as those having a variableregion derived from a murine mAb and a human immunoglobulin constantregion. Such technologies are described in U.S. Pat. Nos. 6,114,598,6,075,181 and 5,877,397 and their respective disclosures, which areherein incorporated by reference in their entirety. Also encompassed bythe present invention is the use of fully humanized monoclonalantibodies, as described in U.S. Pat. No. 6,150,584 and respectivedisclosures, which are herein incorporated by reference in theirentirety.

[0121] Alternatively, techniques described for the production of singlechain antibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA85:5879-5883; and Ward et al., 1989, Nature 341:544-546) can be adaptedto produce single chain antibodies against NHP expression products.Single chain antibodies are formed by linking the heavy and light chainfragments of the Fv region via an amino acid bridge, resulting in asingle chain polypeptide.

[0122] Antibody fragments that recognize specific epitopes may begenerated by known techniques. For example, such fragments include, butare not limited to: F(ab′)₂ fragments, which can be produced by pepsindigestion of an antibody molecule; and Fab fragments, which can begenerated by reducing the disulfide bridges of F(ab′)₂ fragments.Alternatively, Fab expression libraries may be constructed (Huse et al.,1989, Science, 246:1275-1281) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

[0123] Antibodies to a NHP can, in turn, be utilized to generateanti-idiotype antibodies that “mimic” a given NHP, using techniqueswell-known to those skilled in the art (see, e.g., Greenspan and Bona,1993, FASEB J. 7:437-444; and Nisonoff, 1991, J. Immunol.147:2429-2438). For example, antibodies that bind to a NHP domain andcompetitively inhibit the binding of NHP to its cognate receptor can beused to generate anti-idiotypes that “mimic” the NHP and, therefore,bind and activate or neutralize a receptor. Such anti-idiotypicantibodies or fragments of such anti-idiotypes can be used intherapeutic regimens involving a NHP signaling pathway.

[0124] Additionally given the high degree of relatedness of mammalianNHPs, the presently described knock-out mice (having never seen a NHP,and thus never been tolerized to a NHP) have an unique utility, as theycan be advantageously applied to the generation of antibodies againstthe disclosed mammalian NHPs (i.e., a NHP will be immunogenic in NHPknock-out animals).

5.4 Diagosis of NHP-Mediated Disorders

[0125] A variety of methods can be employed for the diagnostic andprognostic evaluation of NHP-mediated disorders. These methods Suchtechniques include, for example, the use of restriction fragment lengthpolymorphisms (RFLPs), which involve sequence variations in one or moreof the recognition sites for any particular restriction enzyme used.

[0126] Additionally, improved methods for analyzing DNA polymorphismsthat can be utilized for the identification of NHP gene mutations havebeen described that capitalize on the presence of variable numbers ofshort, tandemly repeated DNA sequences between certain restrictionenzyme sites. For example, Weber (U.S. Pat. No. 5,075,217, which isincorporated herein by reference in its entirety) describes a DNA markerbased on length polymorphisms in blocks of (dC-dA)_(n)-(dG-dT)_(n) shorttandem repeats. The average separation of (dC-dA)_(n)-(dG-dT)_(n) blocksis estimated to be 30,000-60,000 bp. Markers that are so closely spacedexhibit a high frequency of co-inheritance, and are extremely useful inthe identification of genetic mutations, such as, for example, mutationswithin a NHP gene, and the diagnosis of diseases and disorders relatedto NHP mutations.

[0127] Also, Caskey et al. (U.S. Pat. No. 5,364,759, which isincorporated herein by reference in its entirety) describe a DNAprofiling assay for detecting short tri- and tetra-nucleotide repeatsequences. The process includes extracting the DNA of interest,amplifying the extracted DNA, and labeling the repeat sequences to forma genotypic map of the individual's DNA.

[0128] The level of NHP gene expression can also be assayed by detectingand measuring NHP transcription. For example, RNA from a cell type ortissue known to express, or suspected of expressing, a NHP-gene may beisolated and tested utiizing hybridization or PCR techniques such asthose described herein. The isolated cells can be derived from cellculture or from a patient sample. The analysis of cells taken fromculture may be a necessary step in the assessment of cells to be used aspart of a cell-based gene therapy technique or, alternatively, to testthe effect of compounds on the expression of a NHP gene. Such analysesmay reveal both quantitative and qualitative aspects of the expressionpattern of a NHP gene, including activation or inactivation of NHP geneexpression.

[0129] In one embodiment of such a detection scheme, cDNAs aresynthesized from the RNAs of interest (e.g., by reverse transcription ofthe RNA molecule into cDNA). A sequence within the cDNA is then used asthe template for a nucleic acid amplification reaction, such as a PCRamplification reaction, or the like. For detection of the amplifiedproduct, the nucleic acid amplification may be performed usingradioactively or non-radioactively labeled nucleotides. Alternatively,enough amplified product may be made such that the product may bevisualized by utilizing standard ethidium bromide staining or any othersuitable nucleic acid staining method.

[0130] Additionally, it is possible to perform such NHP gene expressionassays in situ, i.e., directly upon tissue sections (fixed and/orfrozen) of patient tissue obtained from biopsies or resections, suchthat no nucleic acid purification is necessary. Nucleic acid reagentsmay be used as probes and/or primers for such in situ procedures (see,for example, Nuovo, 1992, “PCR In Situ Hybridization: Protocols AndApplications”, Raven Press, N.Y.). Alternatively, if a sufficientquantity of the appropriate cells can be obtained, standard Northernanalysis can be performed to determine the level of mRNA expression of aNHP gene.

[0131] Additionally, NHP oligonucleotide or polynucleotide sequences canbe used as hybridization probes in conjunction with a solid supportmatrix/substrate (e.g., resins, beads, membranes, plastics, polymers,metal or metallized substrates, gene chips, and crystalline orpolycrystalline substrates, etc.).

[0132] 5.4.2 Detection of NHP Gene Products

[0133] Antibodies directed against wild-type or mutant NHP geneproducts, or conserved variants or peptide fragments thereof, which arediscussed above, may also be used in diagnostic and prognostic assays,as described herein. Such diagnostic methods may be used to detectabnormalities in the level of NHP gene expression, or abnormalities inthe structure and/or temporal, tissue, cellular, or subcellular locationof a NHP, and may be performed in vivo or in vitro, such as, forexample, on biopsy tissue.

[0134] For example, antibodies directed to epitopes of a NHP can be usedin vivo to detect the pattern and level of expression of a NHP in thebody. Such antibodies can be labeled, e.g., with a radio-opaque or otherappropriate compound, and injected into a subject, in order to visualizebinding to a NHP expressed in the body, using methods such as X-rays,CAT-scans, or MRI. Labeled antibody fragments, e.g., a Fab or singlechain antibody comprising the smallest portion of the antigen bindingregion, may be preferred for this purpose, to promote crossing theblood-brain barrier and permit labeling of a NHP expressed in the brain.Additionally, any NHP fusion protein or NHP conjugated protein whosepresence can be detected can be administered. For example NHP fusion orconjugated proteins labeled with a radio-opaque or other appropriatecompound can be administered and visualized in vivo, as discussed abovefor labeled antibodies. Further, NHP fusion proteins, such as alkalinephosphatase-NHP or NHP-alkaline phosphatase fusion proteins, can beutilized for in vitro diagnostic procedures.

[0135] Alternatively, immunoassays or fusion protein detection assayscan be utilized on biopsy and autopsy samples in vitro to permitassessment of the expression pattern of a NHP. Such assays can includethe use of antibodies directed to epitopes of any of the domains of aNHP. The use of each or all of these labeled antibodies will yielduseful information regarding translation and intracellular transport ofa NHP, and can identify alterations in processing.

[0136] The tissue or cell type to be analyzed will generally includethose that are known to express, or suspected of expressing, a NHP gene.The protein isolation methods employed herein may, for example, be suchas those previously described (Harlow and Lane, 1988, supra). Theisolated cells can be derived from cell culture or from a patient. Theanalysis of cells taken from culture may be a necessary step in theassessment of cells that could be used as part of a cell-based genetherapy technique or, alternatively, to test the effect of compounds onthe expression of a NHP gene.

[0137] For example, antibodies, or fragments of antibodies, useful inthe present invention may be used to quantitatively or qualitativelydetect the presence of NHP gene products, or conserved variants orpeptide fragments thereof. This can be accomplished, for example, byimmunofluorescence techniques employing a fluorescently labeled antibodycoupled with light microscopic, flow cytometric, or fluorimetricdetection.

[0138] The antibodies (or fragments thereof) or NHP fusion or conjugatedproteins useful in the present invention may, additionally, be employedhistologically, as in immunofluorescence, immunoelectron microscopy ornon-immuno assays, for in situ detection of NHP gene products orconserved variants or peptide fragments thereof. In situ detection maybe accomplished by removing a histological specimen from a patient, andapplying thereto a labeled antibody or fusion protein of the presentinvention. The antibody (or fragment) or fusion protein is preferablyapplied by overlaying the labeled antibody (or fragment) onto abiological sample. Through the use of such a procedure, it is possibleto determine not only the presence of a NHP gene product, or conservedvariants or peptide fragments, but also its distribution in the examinedtissue. Using the present invention, those of ordinary skill willreadily perceive that any of a wide variety of histological methods(such as staining procedures) can be modified in order to achieve suchin situ detection.

[0139] Immunoassays and non-immunoassays for NHP gene products, orconserved variants or peptide fragments thereof, will typically compriseincubating a sample, such as a biological fluid, a tissue extract,freshly harvested cells, or lysates of cells that have been incubated incell culture, in the presence of a detectably labeled antibody capableof identifying NHP gene products, or conserved variants or peptidefragments thereof, and detecting the bound antibody by any of a numberof techniques well-known in the art. The biological sample may bebrought in contact with and immobilized onto a solid phase support orcarrier such as nitrocellulose, or other solid support that is capableof immobilizing cells, cell particles or soluble proteins. The supportmay then be washed with suitable buffers, followed by treatment with thedetectably labeled NHP antibody or NHP fusion protein. The solid phasesupport may then be washed with the buffer a second time to removeunbound antibody or fusion protein. The amount of bound label on solidsupport may then be detected by conventional means.

[0140] The terms “solid phase support or carrier” are intended toinclude any support capable of binding an antigen or an antibody.Well-known supports or carriers include, but are not limited to, glass,polystyrene, polypropylene, polyethylene, dextran, nylon, amylases,natural and modified celluloses, polyacrylamides, gabbros, andmagnetite. The nature of the carrier can be either soluble to someextent or insoluble for the purposes of the present invention. Thesupport material may have virtually any possible structuralconfiguration so long as the coupled molecule is capable of binding toan antigen or antibody. Thus, the support configuration may bespherical, as in a bead, or cylindrical, as in the inside surface of atest tube, or the external surface of a rod. Alternatively, the surfacemay be flat, such as a sheet, test strip, etc. Preferred supportsinclude polystyrene beads. Those skilled in the art will know many othersuitable carriers for binding antibody or antigen, or will be able toascertain the same by use of routine experimentation.

[0141] The binding activity of a given lot of NHP antibody or NHP fusionprotein may be determined according to well-known methods. Those skilledin the art will be able to determine operative and optimal assayconditions for each determination by employing routine experimentation.

[0142] With respect to antibodies, one of the ways in which a NHPantibody can be detectably labeled is by linking the same to an enzymefor use in an enzyme immunoassay (EIA; see, for example, Gosling, ed.,2000, “Immunoassays: A Practical Approach”, Oxford University Press,Inc., N.Y.). The enzyme that is bound to the antibody will react with anappropriate substrate, preferably a chromogenic substrate, in such amanner as to produce a chemical moiety that can be detected, forexample, by spectrophotometric, fluorimetric or visual means. Enzymesthat can be used to detectably label the antibody include, but are notlimited to, malate dehydrogenase, staphylococcal nuclease,delta-5-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. The detection can be accomplished by colorimetricmethods that employ a chromogenic substrate for the enzyme. Detectionmay also be accomplished by visual comparison of the extent of enzymaticreaction of a substrate in comparison with similarly prepared standards.

[0143] Additionally, detection may also be accomplished using any of avariety of other immunoassays. For example, by radioactively labelingthe antibodies or antibody fragments, it is possible to detect NHPsthrough the use of a radioimmunoassay (RIA). The radioactive isotope canbe detected by such means as the use of a gamma or scintillationcounter, or by autoradiography.

[0144] It is also possible to label the antibody with a fluorescentcompound. When the fluorescently labeled antibody is exposed to light ofthe proper wavelength, its presence can then be detected due tofluorescence. Exemplary fluorescent labeling compounds include, but arenot limited to, fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine. Theantibody can also be detectably labeled using fluorescence emittingmetals such as ¹⁵²Eu, or others of the lanthanide series. These metalscan be attached to the antibody using such metal chelating groups asdiethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraaceticacid (EDTA). The antibody also can be detectably labeled by coupling itto a chemiluminescent compound. The presence of thechemiluminescent-tagged antibody is then determined by detecting thepresence of luminescence that arises during the course of a chemicalreaction. Examples of particularly useful chemiluminescent labelingcompounds include, but are not limited to, luminol, isoluminol,theromatic acridinium ester, imidazole, acridinium salt and oxalateester.

[0145] Likewise, a bioluminescent compound may be used to label the NHPantibodies of the present invention. Bioluminescence is a type ofchemiluminescence found in biological systems, in which a catalyticprotein increases the efficiency of the chemiluminescent reaction. Thepresence of a bioluminescent protein is determined by detecting thepresence of luminescence. Exemplary bioluminescent compounds forpurposes of labeling include, but are not limited to, luciferin,luciferase and aequorin (green fluorescent protein; as described in U.S.Pat. Nos. 5,491,084, 5,625,048, 5,777,079, 5,795,737, 5,804,387,5,874,304, 5,968,750, 5,976,796, 6,020,192, 6,027,881, 6,054,321,6,096,865, 6,146,826, 6,172,188 and 6,265,548, each of which is herebyincorporated by: reference).

5.5 Screening Assays for Compounds that Modulate NHP Expression orActivity

[0146] The following assays are designed to identify compounds thatinteract with (e.g., bind to) NHPs, compounds that interact with (e.g.,bind to) intracellular proteins that interact with NHPs, compounds thatinteract with (e.g., bind to) both intracellular and extracellularproteins or receptors that regulate NHP activity and expression,compounds that interfere with the interaction of NHPs or proteins orcompounds involved in NHP-mediated activity, and compounds that modulatethe activity of a NHP gene (i.e., modulate the level of NHP geneexpression) or modulate the level of NHPs. Assays may additionally beutilized that identify compounds that bind to NHP gene regulatorysequences (e.g., promoter sequences) and that may modulate NHP geneexpression.

[0147] The compounds that can be screened in accordance with the presentinvention include, but are not limited to, peptides, antibodies andfragments thereof, and other organic compounds (e.g., peptidomimetics,small organic compounds) that bind to a NHP and either mimic or increasethe activity of a NHP (i.e., agonists) or inhibit the activity of a NHP(i.e., antagonists); as well as peptides, antibodies or fragmentsthereof, and other organic compounds that mimic or increase NHP activityor inhibit the activity of a NHP.

[0148] Such compounds may include, but are not limited to, peptides suchas, for example, soluble peptides, including, but not limited to,members of random peptide libraries (see, e.g., Lam et al., 1991, Nature354:82-84; Houghten et al., 1991, Nature 354:84-86), and combinatorialchemistry-derived molecular libraries made of D- and/or L-configurationamino acids, phosphopeptides (including, but not limited to, members ofrandom or partially degenerate, directed phosphopeptide libraries; see,e.g., Songyang et al., 1993, Cell 72:767-778), antibodies (including,but not limited to, polyclonal, monoclonal, humanized, anti-idiotypic,chimeric or single chain antibodies, and Fab, F(ab′)₂ and Fab expressionlibrary fragments, and epitope-binding fragments thereof), and smallorganic or inorganic molecules.

[0149] Other compounds that can be screened in accordance with theinvention include, but are not limited to, small organic molecules thatare able to gain entry into an appropriate cell and affect theexpression of a NHP gene, or some other gene involved in a NHP pathway(e.g., by interacting with the regulatory region or transcriptionfactors involved in gene expression); or such compounds that affect theactivity of NHPs or the activity of some other intracellular factorinvolved in a NHP pathway.

[0150] Computer modeling and searching technologies permitidentification of compounds, or the improvement of already identifiedcompounds, that can modulate NHP expression or activity. Havingidentified such a compound or composition, the active sites or regionsare identified. The active site can be identified using methods known inthe art including, for example, from the amino acid sequences ofpeptides, from the nucleotide sequences of nucleic acids, or from studyof complexes of the relevant compound or composition with its naturalligand. In the latter case, chemical or X-ray crystallographic methodscan be used to find the active site by finding where on the factor thecomplexed ligand is found. Next, the three dimensional geometricstructure of the active site is determined. This can be done by knownmethods, including X-ray crystallography, which can determine a completemolecular structure. On the other hand, solid or liquid phase NMR can beused to determine certain intra-molecular distances. Any otherexperimental method of structure determination can be used to obtainpartial or complete geometric structures. The geometric structures maybe measured with a complexed ligand, natural or artificial, which mayincrease the accuracy of the active site structure determined.

[0151] If an incomplete or insufficiently accurate structure isdetermined, the methods of computer based numerical modeling can be usedto complete the structure or improve its accuracy. Any recognizedmodeling method can be used, including parameterized models specific toparticular biopolymers such as proteins or nucleic acids, moleculardynamics models based on computing molecular motions, statisticalmechanics models based on thermal ensembles, or combined models. Formost types of models, standard molecular force fields, representing theforces between constituent atoms and groups, are necessary, and can beselected from force fields known in physical chemistry. The incompleteor less accurate experimental structures can serve as constraints on thecomplete and more accurate structures computed by these modelingmethods.

[0152] Finally, having determined the structure of the active site,either experimentally, by modeling, or a combination thereof, candidatemodulating compounds can be identified by searching databases containingcompounds along with information on their molecular structure. Such asearch seeks compounds having structures that match the determinedactive site structure and that interact with the groups defining theactive site. Such a search can be manual, but is preferably computerassisted. The compounds found from such a search are potential NHPmodulating compounds.

[0153] Alternatively, these methods can be used to identify improvedmodulating compounds from an already known modulating compound orligand. The composition of the known compound can be modified and thestructural effects of modification can be determined using theexperimental and computer modeling methods described above applied tothe new composition. The altered structure is then compared to theactive site structure of the compound to determine if an improved fit orinteraction results. In this manner systematic variations incomposition, such as by varying side groups, can be quickly evaluated toobtain modified modulating compounds or ligands of improved specificityor activity.

[0154] Further experimental and computer modeling methods useful toidentify modulating compounds based upon identification of the activesites of NHPs, and related transduction and transcription factors, willbe apparent to those of skill in the art.

[0155] Examples of molecular modeling systems are the CHARMM and QUANTAprograms (Polygen Corporation, Waltham, Mass.). CHARMM performs theenergy minimization and molecular dynamics functions. QUANTA performsthe construction, graphic modeling and analysis of molecular structure.QUANTA allows interactive construction, modification, visualization, andanalysis of the behavior of molecules with each other.

[0156] A number of articles review computer modeling of drugsinteractive with specific proteins, such as Rotivinen et al., 1988, ActaPharmaceutical Fennica 97:159-166; Ripka, New scientist 54-57 (Jun. 16,1988); McKinaly and Rossmann, 1989, Ann. Rev. Pharmacol. Toxiciol.29:111-122; Perry and Davies, OSAR: Quantitative Structure-ActivityRelationships in Drug Design, pp. 189-193 (Alan R. Liss, Inc. 1989);Lewis and Dean, 1989, Proc. R. Soc. Lond. 236:125-140 and 141-162; and,with respect to a model receptor for nucleic acid components, Askew etal., 1989, J. Am. Chem. Soc. 111:1082-1090. Other computer programs thatscreen and graphically depict chemicals are available from companiessuch as BioDesign, Inc. (Pasadena, Calif.), Allelix, Inc. (Mississauga,Ontario, Canada), and Hypercube, Inc. (Cambridge, Ontario). Althoughthese are primarily designed for application to drugs specific toparticular proteins, they can be adapted to the design of drugs specificto regions of DNA or RNA, once that region is identified.

[0157] Although described above with reference to design and generationof compounds that could alter binding, one could also screen librariesof known compounds, including natural products or synthetic chemicals,and biologically active materials, including proteins, for compoundsthat are inhibitors or activators of NHPs.

[0158] Compounds identified via assays such as those described hereinmay be useful, for example, in further elaborating the biologicalfunction of a NHP gene product, and for ameliorating NHP-relateddisorders.

[0159] 5.5.1 In Vitro Screening Assays for Compounds that Bind to a NHP

[0160] In vitro systems may be designed to identify compounds capable ofinteracting with (e.g., binding to) NHPs. The compounds thus identified(such as NHP modulators, natural NHP substrates, etc.) can be useful,for example, in modulating the activity of wild-type and/or mutant NHPgene products; in elaborating the biological function of NHPs; inscreens for identifying compounds that disrupt normal NHP interactions;or in themselves directly disrupt such interactions.

[0161] The principle of the assays used to identify compounds that bindto a NHP involves preparing a reaction mixture of a NHP and the testcompound under conditions and for a time sufficient to allow the twocomponents to interact and bind, thus forming a complex that can beremoved and/or detected in the reaction mixture. The NHP species usedcan vary depending upon the goal of the screening assay. For example,where agonists of the natural ligand/substrate are sought, full lengthNHPs, or a soluble truncated NHP polypeptide that affords advantages inthe assay system (e.g., labeling, isolation of the resulting complex,etc.) can be utilized.

[0162] The screening assays can be conducted in a variety of ways. Forexample, one method to conduct such an assay would involve anchoring aNHP protein, polypeptide, peptide, or fusion protein, or the testsubstance, onto a solid phase and detecting NHP/test compound complexesanchored on the solid phase at the end of the reaction. In oneembodiment of such a method, the NHP reactant may be anchored onto asolid surface, and the test compound, which is not anchored, may belabeled, either directly or indirectly. Examples of some of thetechnologies available to immobilize the molecules are discussed inCass, ed., “Immobilized Biomolecules In Analysis: A Practical Approach”,Oxford University Press, N.Y.

[0163] In practice, microtiter plates may conveniently be utilized asthe solid phase. The anchored component may be immobilized bynon-covalent or covalent attachments. Non-covalent attachment may beaccomplished by simply coating the solid surface with a solution of theprotein and drying. Alternatively, an immobilized antibody, preferably amonoclonal antibody, specific for the protein to be immobilized may beused to anchor the protein to the solid surface. The surfaces may beprepared in advance and stored. molecular structure. Such a search seekscompounds having structures that match the determined active sitestructure and that interact with the groups defining the active site.Such a search can be manual, but is preferably computer assisted. Thecompounds found from such a search are potential NHP modulatingcompounds.

[0164] Alternatively, these methods can be used to identify improvedmodulating compounds from an already known modulating compound orligand. The composition of the known compound can be modified and thestructural effects of modification can be determined using theexperimental and computer modeling methods described above applied tothe new composition. The altered structure is then compared to theactive site structure of the compound to determine if an improved fit orinteraction results. In this manner systematic variations incomposition, such as by varying side groups, can be quickly evaluated toobtain modified modulating compounds or ligands of improved specificityor activity.

[0165] Further experimental and computer modeling methods useful toidentify modulating compounds based upon identification of the activesites of NHPs, and related transduction and transcription factors, willbe apparent to those of skill in the art.

[0166] Examples of molecular modeling systems are the CHARMM and QUANTAprograms (Polygen Corporation, Waltham, Mass.). CHARMM performs theenergy minimization and molecular dynamics functions. QUANTA performsthe construction, graphic modeling and analysis of molecular structure.QUANTA allows interactive construction, modification, visualization, andanalysis of the behavior of molecules with each other.

[0167] A number of articles review computer modeling of drugsinteractive with specific proteins, such as Rotivinen et al., 1988, ActaPharmaceutical Fennica 97:159-166; Ripka, New Scientist 54-57 (Jun. 16,1988); McKinlay and Rossmann, 1989, Ann. Rev. Pharmacol. Toxiciol.29:111-122; Perry and Davies, OSAR: employed for identifying proteinsthat interact with NHPS. Among the traditional methods that may beemployed are co-immunoprecipitation, crosslinking and co-purificationthrough gradients or chromatographic columns of cell lysates, orproteins obtained from cell lysates, and a NHP to identify proteins inthe lysate that interact with NHPs. For these assays, the NHP componentused can be a full length NHP, a peptide or polypeptide corresponding toone or more domains of a NHP, or a fusion protein containing one or moredomains of a NHP. Once isolated, such an intracellular protein can beidentified and can, in turn, be used in conjunction with standardtechniques to identify proteins with which it interacts. For example, atleast a portion of the amino acid sequence of an intracellular proteinthat interacts with a NHP can be ascertained using techniques well-knownto those of skill in the art, such as via the Edman degradationtechnique (see, e.g., Creighton, 1983, supra, pp.34-49). The amino acidsequence obtained may be used as a guide for the generation ofoligonucleotide mixtures that can be used to screen for gene sequencesencoding such intracellular proteins. Screening may be accomplished, forexample, by standard hybridization or PCR techniques. Techniques for thegeneration of oligonucleotide mixtures and screening are well-known(see, e.g., Ausubel, supra., and Innis et al., eds. “PCR Protocols: AGuide to Methods and Applications”, 1990, Academic Press, Inc., N.Y.).

[0168] Additionally, methods may be employed that result in thesimultaneous identification of genes that encode proteins that arecapable of interacting with NHPs. These methods include, for example,probing expression libraries, in a manner similar to the well-knowntechnique of antibody probing of lambda gt11 libraries, using a labeledNHP protein, polypeptide, peptide or fusion protein, e.g., a NHPpolypeptide or NHP domain fused to a marker (e.g., an enzyme, fluor,luminescent protein, or dye), or an Ig-Fc domain.

[0169] One method that detects protein interactions in vivo, thetwo-hybrid system, is described in detail for illustration only and notby way of limitation. One version of this system utilizes yeast cells(Chien et al., 1991, Proc. Natl. Acad. Sci. USA, 88:9578-9582), whileanother uses mammalian cells (Luo et al., 1997, Biotechniques22:350-352). Both the yeast and mammalian two-hybrid systems arecommercially available from Clontech (Palo Alto, Calif.), and arefurther described in U.S. Pat. Nos. 5,283,173, 5,468,614, and 5,667,973,which are herein incorporated by reference in their entirety.

[0170] Briefly, utilizing such a system, plasmids are constructed thatencode two hybrid proteins: one plasmid consists of nucleotides encodingthe DNA-binding domain of a transcription activator protein fused to aNHP nucleotide sequence encoding a NHP protein, polypeptide, peptide orfusion protein, and the other plasmid consists of nucleotides encodingan activation domain of a transcription activator protein fused to acDNA encoding an unknown protein to be tested for interaction with aNHP, which has been recombined into this plasmid as part of a cDNAlibrary. The DNA-binding domain fusion plasmid and the cDNA library aretransformed into a strain of the yeast Saccharomyces cerevisiae or amammalian cell (such as Saos-2, CHO, CV1, Jurkat or HeLa) that containsa reporter gene (e.g., HBS, lacZ, CAT, or a gene encoding an essentialamino acid synthetase) whose regulatory region contains the binding siteof the transcription activator. Either hybrid protein alone cannotactivate transcription of the reporter gene: the DNA-binding domainhybrid cannot because it does not provide activation function; and theactivation domain hybrid cannot because it cannot localize to thebinding site of the activator. Interaction of the two hybrid proteinsreconstitutes the functional activator protein and results in expressionof the reporter gene, which is detected by an assay for the reportergene product.

[0171] The two-hybrid system or related methodology may be used toscreen activation domain libraries for proteins that interact with the“bait” gene product. By way of example, and not by way of limitation, aNHP may be used as the bait gene product. Total genomic or cDNAsequences are fused to DNA encoding an activation domain. This libraryand a plasmid encoding a hybrid of a bait NHP gene product fused to theDNA-binding domain are co-transformed into a reporter strain, and theresulting transformants are screened for those that express the reportergene. For example, and not by way of limitation, a bait NHP sequence,such as an open reading frame of a NHP (or a domain of a NHP) can becloned into a vector such that it is translationally fused to DNAencoding the DNA-binding domain of the GAL4 protein. These colonies arepurified and the library plasmids responsible for reporter geneexpression are isolated. DNA sequencing is then used to identify theproteins encoded by the library plasmids.

[0172] A cDNA library of the cell line from which proteins that interactwith a bait NHP gene product are to be detected can be made usingmethods routinely practiced in the art. According to one particularsystem, for example, the cDNA fragments can be inserted into a vectorsuch that they are translationally fused to the transcriptionalactivation domain of GAL4. This library can be co-transformed along withthe bait NHP gene-GAL4 fusion plasmid into a yeast strain that cannotgrow without added histidine, and that contains a HIS3 gene driven by apromoter that contains GAL4 activation sequence. A cDNA encoded protein,fused to GAL4 transcriptional activation domain, which interacts withthe bait NHP gene product will reconstitute an active GAL4 protein andthereby drive expression of the HIS3 gene. Colonies that express HIS3can be detected by their growth on petri dishes containing semi-solidagar based media lacking histidine. The cDNA can then be purified fromthese strains, and used to produce and isolate the bait NHPgene-interacting protein using techniques routinely practiced in theart.

[0173] 5.5.3 Assays for Compounds that Interfere with NHP Activity

[0174] The macromolecules that interact with NHPs are referred to, forpurposes of this discussion, as “binding partners”. These bindingpartners are likely to be involved in a NHP pathway, and therefore, mayhave a role in NHP-mediated disorders. Therefore, it is desirable toidentify compounds that interfere with or disrupt the interaction ofsuch binding partners with NHPs, and that may be useful in regulatingthe activity of NHPs and controlling NHP-mediated disorders.

[0175] The basic principle of the assay systems used to identifycompounds that interfere with the interaction between a NHP and itsbinding partner or partners involves preparing a reaction mixturecontaining a NHP protein, polypeptide, peptide or fusion protein, andthe binding partner under conditions and for a time sufficient to allowthe components to interact and bind, thus forming a complex. In order totest a compound for inhibitory activity, the reaction mixture isprepared in the presence and absence of the test compound. The testcompound may be initially included in the reaction mixture, or may beadded at a time subsequent to the addition of the NHP moiety and itsbinding partner(s). Control reaction mixtures are incubated without thetest compound or with a placebo. The formation of complexes between theNHP moiety and the binding partner is then detected. The formation of acomplex in the control reaction, but not in the reaction mixturecontaining the test compound, indicates that the compound interfereswith the interaction of the NHP and the interactive binding partner.Additionally, complex formation within reaction mixtures containing thetest compound and a normal NHP protein may also be compared to complexformation within reaction mixtures containing the test compound and amutant NHP. This comparison may be important in those cases wherein itis desirable to identify compounds that disrupt interactions of mutantbut not normal NHPs.

[0176] Assays for compounds that interfere with the interaction of NHPsand binding partner(s) can be conducted in a heterogeneous orhomogeneous format. Heterogeneous assays involve anchoring either theNHP moiety product or the binding partner onto a solid phase anddetecting complexes anchored on the solid phase at the end of thereaction. In homogeneous assays, the entire reaction is carried out in aliquid phase. In either approach, the order of addition of reactants canbe varied to obtain different information about the compounds beingtested. For example, test compounds that interfere with the interactionby competition can be identified by conducting the reaction in thepresence of the test substance, i.e., by adding the test substance tothe reaction mixture prior to or simultaneously with the NHP moiety andinteractive binding partner. Alternatively, test compounds that disruptpreformed complexes, e.g., compounds with higher binding constants thatdisplace one of the components from the complex, can be tested by addingthe test compound to the reaction mixture after complexes have beenformed. The various formats are described briefly below.

[0177] In a heterogeneous assay system, either the NHP moiety or theinteractive binding partner is anchored onto a solid surface, while thenon-anchored species is labeled, either directly or indirectly. Inpractice, microtiter plates are conveniently utilized. The anchoredspecies may be immobilized by non-covalent or covalent attachments.Non-covalent attachment may be accomplished simply by coating the solidsurface with a solution of a NHP gene product or binding partner anddrying. Alternatively, an immobilized antibody specific for the speciesto be anchored may be used to anchor the species to the solid surface.The surfaces may be prepared in advance and stored.

[0178] In order to conduct the assay, the partner of the immobilizedspecies is exposed to the coated surface with or without the testcompound. After the reaction is complete, unreacted components areremoved (e.g., by washing), and any complexes formed will remainimmobilized on the solid surface. The detection of complexes anchored onthe solid surface can be accomplished in a number of ways. Where thenon-immobilized species is pre-labeled, the detection of labelimmobilized on the surface indicates that complexes were formed. Wherethe non-immobilized species is not pre-labeled, an indirect label can beused to detect complexes anchored on the surface, e.g., using a labeledantibody specific for the initially non-immobilized species (theantibody, in turn, may be directly labeled or indirectly labeled with alabeled anti-Ig antibody). Depending upon the order of addition ofreaction components, test compounds that inhibit complex formation orthat disrupt preformed complexes can be detected.

[0179] Alternatively, the reaction can be conducted in a liquid phase inthe presence or absence of the test compound, the reaction productsseparated from unreacted components, and complexes detected, e.g., usingan immobilized antibody specific for one of the binding components toanchor any complexes formed in solution, and a labeled antibody specificfor the other partner to detect anchored complexes. Again, dependingupon the order of addition of reactants to the liquid phase, testcompounds that inhibit complex or that disrupt preformed complexes canbe identified.

[0180] In an alternate embodiment of the invention, a homogeneous assaycan be used. In this approach, a preformed complex of a NHP moiety andthe interactive binding partner is prepared in which either the NHP orits binding partner is labeled, but the signal generated by the label isquenched due to formation of the complex (see, e.g., U.S. Pat. No.4,109,496, incorporated herein by reference, which utilizes thisapproach for immunoassays). The addition of a test substance thatcompetes with and displaces one of the species from the preformedcomplex will result in the generation of a signal above background. Inthis way, test substances that disrupt NHP/intracellular binding partnerinteractions can be identified.

[0181] In a particular embodiment, a NHP fusion protein can be preparedfor immobilization. For example, a NHP, or a peptide fragment thereof,e.g., corresponding to one or more particular domain(s), can be fused toglutathione-S-transferase (GST) using a fusion vector, such aspGEX-5X-1, in such a manner that the GST binding activity is maintainedin the resulting fusion protein. An interactive binding partner,identified as described herein, can be purified and used to raisepolyclonal and monoclonal antibodies, using methods routinely practicedin the art. Such antibodies can be labeled with a radioactive isotope,¹²⁵I for example, by methods routinely practiced in the art. In aheterogeneous assay, such GST-NHP fusion proteins can be anchored toglutathione-agarose beads. The interactive binding partner can then beadded in the presence or absence of the test compound in a manner thatallows interaction and binding to occur. At the end of the reactionperiod, unbound material can be washed away, and a labeled monoclonalantibody that binds the binding partner can be added to the system andallowed to bind to complexed binding partner. The interaction betweenthe NHP and the interactive binding partner can be detected by measuringthe amount of radioactivity that remains associated with theglutathione-agarose beads. A successful inhibition of the interaction bythe test compound will result in a decrease in measured radioactivity.

[0182] Alternatively, a GST-NHP fusion protein and an interactivebinding partner can be mixed together in liquid in the absence of thesolid glutathione-agarose beads. The test compound can be added eitherduring or after the species are allowed to interact. This mixture canthen be added to the glutathione-agarose beads and unbound material iswashed away. Again the extent of inhibition of the NHP/binding partnerinteraction can be detected by adding a labeled antibody against thebinding partner and measuring the radioactivity associated with thebeads.

[0183] In another embodiment of the invention, where the binding partneris a protein, these same techniques can be employed using peptidefragments that correspond to one or more of the binding domains of a NHPand/or the interactive binding partner, in place of one or both of thefull length proteins. Any number of methods routinely practiced in theart can be used to identify and isolate the binding domains or regions.These methods include, but are not limited to, mutagenesis of the geneencoding one of the proteins, and screening for disruption of binding ina co-immunoprecipitation assay. Compensating mutation(s) in the sequenceencoding the second species in the complex can then be selected.Sequence analysis of the sequences encoding the respective proteins willreveal the mutation(s) that correspond to the region of the proteininvolved in interactive binding. Alternatively, one protein can beanchored to a solid surface using methods described above, and allowedto interact with and bind to its labeled binding partner, which has beentreated with a proteolytic enzyme, such as trypsin. After washing, ashort, labeled peptide comprising the binding domain may remainassociated with the solid material, which can be isolated and identifiedby amino acid sequencing. Also, once a sequence encoding the bindingpartner is obtained, short polynucleotide segments can be engineered toexpress peptide fragments of the protein, which can then be tested forbinding activity, and purified or synthesized.

[0184] For example, and not by way of limitation, a NHP protein,polypeptide or peptide can be anchored to a solid material, as describedabove, by making a GST-NHP fusion protein and allowing it to bind toglutathione agarose beads. The interactive binding partner can belabeled with a radioactive isotope, such as ³⁵S, and cleaved with aproteolytic enzyme, such as trypsin. Cleavage products can then be addedto the anchored GST-NHP fusion protein and allowed to bind. Afterwashing away unbound peptides, labeled bound material, representing theintracellular binding partner binding domain, can be eluted, purified,and analyzed to determine the amino acid sequence by well-known methods.Peptides so identified can be produced synthetically or fused toappropriate facilitative proteins using recombinant DNA technology.

[0185] 5.5.4 The Use of Compounds to Treat NHP-Mediated Disorders

[0186] The invention also encompasses the use of agonists andantagonists of a NHP (including small molecules and large molecules),mutant versions of a NHP or portions thereof that compete with nativeNHPs, peptides, and antibodies, as well as nucleotide sequences that canbe used to inhibit the expression of a NHP (e.g., antisense and ribozymemolecules, and gene or regulatory sequence replacement constructs) or toenhance the expression of NHP polynucleotides (e.g., expressionconstructs that place the described polynucleotide under the control ofa strong promoter system) in the treatment of NHP-mediated disorders.Compounds including, but not limited to, those identified via assaytechniques such as those described above, can be tested for the abilityto ameliorate symptoms associated with NHP-mediated disorders.

[0187] The assays described above can identify compounds that affect NHPactivity, or compounds that affect NHP gene activity (by affecting NHPgene expression, including molecules, e.g., proteins or small organicmolecules, that affect or interfere with splicing events so thatexpression of a full length or a truncated form of a NHP can bemodulated). However, it should be noted that the assays described canalso be used to identify compounds that indirectly modulate NHPs. Theidentification and use of compounds that affect a NHP-independent stepin a NHP pathway are also within the scope of the invention. Compoundsthat indirectly affect NHP activity can also be used as part of atherapeutic method for the treatment of NHP-mediated disorders.

[0188] The invention additionally encompasses cell-based and animalmodel-based assays for the identification of compounds exhibiting anability to ameliorate the symptoms of NHP-mediated disorders. Cell-basedsystems used to identify compounds that may act to ameliorateNHP-mediated disorder symptoms can include, for example, recombinant ornon-recombinant cells, such as cell lines that express a NHP sequence.Host cells (e.g., COS cells, CHO cells, fibroblasts) geneticallyengineered to express a functional NHP can also be used. The presence ofa functional NHP can be determined, for example, by a chemical or aphenotypic change, the induction of another host cell gene, a change inion flux (e.g., Ca⁺⁺), or tyrosine phosphorylation of host cellproteins, etc.

[0189] In utilizing such cell systems, cells may be exposed to acompound suspected of exhibiting an ability to ameliorate the symptomsof NHP-mediated disorders, at a sufficient concentration and for a timesufficient to elicit such an amelioration of the symptoms ofNHP-mediated disorders in the exposed cells. After exposure, the cellscan be assayed to measure alterations in NHP expression, e.g., byassaying cell lysates for NHP mRNA transcripts (e.g., by Northernanalysis or RT-PCR), or by assaying for the level of a NHP proteinexpressed in the cell (e.g., by SDS-PAGE and Western blot orimmunoprecipitation); compounds that regulate or modulate NHP expressionare good candidates as therapeutics. Alternatively, the cells can beexamined to determine whether one or more NHP disorder-like cellularphenotype has been altered to resemble a more normal or more wild-type,non-NHP disorder phenotype, or a phenotype more likely to produce alower incidence or severity of disorder symptoms. Still further, theexpression and/or activity of components of the signal transductionpathway(s) of which a NHP is a part, or the activity of a NHP signaltransduction pathway itself, can be assayed.

[0190] In addition, animal-based NHP-mediated disorder systems may beused to identify compounds capable of treating or ameliorating symptomsassociated with NHP-mediated disorders. These animals may be transgenic,knockout, or knock-in (preferably humanized knock-ins where, forexample, the endogenous animal NHP gene has been replaced by a human NHPsequence) animals, as described herein. Such animal models may be usedas test substrates for the identification of drugs, pharmaceuticals,therapies and interventions that may be effective in treating suchdisorders. For example, animal models can be exposed to a compoundsuspected of exhibiting an ability to ameliorate symptoms ofNHP-mediated disorders, at a sufficient concentration and for a timesufficient to elicit such an amelioration of NHP disorder associatedsymptoms in the exposed animals. The response of the animals to theexposure may be monitored by assessing the reversal of symptomsassociated with NHP-mediated disorders. With regard to intervention, anytreatments that reverse, halt or slow the progression of any aspect ofsymptoms associated with NHP disorders should be considered ascandidates for therapeutic intervention in treatment of human NHPdisorders. Dosages of test agents may be determined by deriving toxicityand dose-response curves.

5.6 Pharmaceutical Preparations and Methods of Administration

[0191] Compounds that are determined to affect expression of thesequences of the current invention, or the interaction of the peptidesand proteins of the present invention with any of their bindingpartners, can be administered to a patient at therapeutically effectivedoses to treat or ameliorate low white blood cell counts. Atherapeutically effective dose refers to that amount of the compoundsufficient to result in any delay in onset, amelioration or retardationof disease symptoms.

[0192] 5.6.1 Effective Dose

[0193] Toxicity and therapeutic efficacy of such compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD₅₀ (the dose lethal to50% of the population) and the ED₅₀ (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index, and it can be expressed as the ratioLD₅₀/ED₅₀. Compounds that exhibit large therapeutic indices arepreferred. Compounds that exhibit toxic side effects may be used incertain embodiments, however care should usually be taken to designdelivery systems that target such compounds preferentially to the siteof affected tissue, in order to minimize potential damage to uninfectedcells and, thereby, reduce side effects.

[0194] The data obtained from cell culture assays and animal studies canbe used in formulating a range of dosages for use in humans. The dosagesof such compounds lie preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the methods of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound that achieves a half maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

[0195] When the therapeutic treatment of disease is contemplated, theappropriate dosage may also be determined using animal studies todetermine the maximal tolerable dose, or MTD, of a bioactive agent perkilogram weight of the test subject. In general, at least one animalspecies tested is mammalian. Those skilled in the art regularlyextrapolate doses for efficacy and avoiding toxicity to other species,including human. Before human studies of efficacy are undertaken, PhaseI clinical studies in normal subjects help establish safe doses.

[0196] Additionally, the bioactive agent may be complexed with a varietyof well established compounds or structures that, for instance, enhancethe stability of the bioactive agent, or otherwise enhance itspharmacological properties (e.g., increase in vivo half-life, reducetoxicity, etc.).

[0197] The therapeutic agents will be administered by any number ofmethods known to those of ordinary skill in the art including, but notlimited to, inhalation; subcutaneous (sub-q); intravenous (I.V.);intraperitoneal (I.P.); intramuscular (I.M.), or intrathecal injection;or topically applied (transderm, ointments, creams, salves, eye drops,and the like).

[0198] 5.6.2 Formulations and Use

[0199] Pharmaceutical compositions for use in accordance with thepresent invention may be formulated in conventional manners using one ormore physiologically acceptable carriers or excipients.

[0200] Thus, the compounds and their physiologically acceptable saltsand solvates may be formulated for administration by inhalation orinsufflation (either through the mouth or the nose), or oral, buccal,parenteral or rectal administration.

[0201] For oral administration, the pharmaceutical compositions may takethe form of, for example, tablets or capsules prepared by conventionalmeans with pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets may be coated by methods well-known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoringagents, coloring agents and sweetening agents as appropriate.

[0202] Preparations for oral administration may be suitably formulatedto give controlled release of the active compound. For buccaladministration the compositions may take the form of tablets or lozengesformulated in conventional manner.

[0203] For administration by inhalation, the compounds for use accordingto the present invention are conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol, the dosageunit may be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, e.g., gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

[0204] The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

[0205] The compounds may also be formulated as compositions for rectaladministration such as suppositories or retention enemas, e.g.,containing conventional suppository bases such as cocoa butter or otherglycerides.

[0206] In addition to the formulations described previously, thecompounds may also be formulated as a depot preparation. Such longacting formulations may be administered by implantation (for examplesubcutaneously or intramuscularly) or by intramuscular injection. Thus,for example, the compounds may be formulated with suitable polymeric orhydrophobic materials (for example as an emulsion in an acceptable oil),ion exchange resins, or as sparingly soluble derivatives, for example,as a sparingly soluble salt. The compositions may, if desired, bepresented in a pack or dispenser device, which may contain one or moreunit dosage forms containing the active ingredient. The pack may forexample comprise metal or plastic foil, such as a blister pack. The packor dispenser device may be accompanied by instructions foradministration.

[0207] The examples below are provided to illustrate the subjectinvention. These examples are provided by way of illustration only, andare not included for the purpose of limiting the invention in any waywhatsoever.

6.0 EXAMPLES 6.1 NHP Gene Disrupted Mice

[0208] Gene trapping is a method of nonspecific insertional mutagenesisthat uses a fragment of DNA coding for a reporter or selectable markergene as a mutagen. Gene trap vectors have been designed to integrateinto introns or exons in a manner that allows the cellular splicingmachinery to splice vector encoded exons to cellular mRNAs. Commonly,gene trap vectors contain selectable marker sequences that are precededby strong splice acceptor sequences and are not preceded by a promoter.Thus, when such vectors integrate into a gene, the cellular splicingmachinery splices exons from the trapped gene onto the 5′ end of theselectable marker sequence. Typically, such selectable marker genes canonly be expressed if the vector encoding the gene has integrated into anintron. The resulting gene trap events are subsequently identified byselecting for cells that can survive selective culture.

[0209] Embryonic stem cells (Lex-1 cells derived from murine strain129SvEv^(Brd)) were mutated by a process involving the insertion of atleast a portion of a genetically engineered vector sequence into the NHPgene. The mutated embryonic stem cells were then microinjected intoblastocysts, which were subsequently introduced into pseudopregnantfemale hosts and carried to term using established methods, such asthose described in, for example, Zambrowicz et al., eds., “MouseMutagenesis”, 1998, Lexicon Press, The Woodlands, Tex., and periodicupdates thereof, herein incorporated by reference. In this case, thevirus inserted in the forward orientation, close to the nucleotidescoding for amino acid 135, and disrupted the NHP gene. The resultingchimeric animals were subsequently bred to produce offspring capable ofgermline transmission of an allele containing the engineered mutation inthe NHP gene.

[0210] Techniques useful to disrupt a gene in a cell, and especially anES cell, that may already have a disrupted gene are disclosed in U.S.Pat. Nos. 6,136,566, 6,139,833 and 6,207,371, and U.S. patentapplication Ser. No. 08/728,963, each of which are hereby incorporatedherein by reference in their entirety.

[0211] 6.1.1 The effect of NHP Disruption on Mouse Physiology

[0212] The genetic distribution of wild-type (11), heterozygous (28) andhomozygous (19) animals appears to be normal. Thus, for the remainingstudies, mice homozygous for the disruption of the NHP gene were studiedin conjunction with heterozygous and wild-type litter mates. During thisanalysis, the mice were subjected to a medical work-up using anintegrated suite of medical diagnostic procedures designed to assess thefunction of the major mammalian organ systems in the subject. Bystudying numerous mice in which the NHP gene had been disrupted, inconjunction with wild-type litter mates, more reliable and repeatabledata was obtained. Disruption of the NHP gene resulted in, among othereffects, an unexpected increase in white blood cell counts, as describedin greater detail below. The disruption of the NHP gene was confirmed byRT-PCR.

[0213] Additional studies of the expression patterns of human and murineNHPs showed that the NHPs can be detected in certain mouse and humantissues by RT-PCR. NHP transcripts were detected in mouse tissue derivedfrom mouse brain, thymus, and heart. NHP transcripts were detected inhuman tissue derived from human prostate and testis.

[0214] 6.1.2 Size, Percent Body Fat, and Bone Mineral Density of NHPKnockouts

[0215] Body composition and percent body fat was measured by dual energyX-ray absorptiometry (DEXA) using the Piximus small animal densitometer(Lunar Corporation, Madison, Wis.). Individual mice were sedated withAvertin (1.25% solution, 2.5 mg/10 gm body weight delivered byintraperitoneal injection), immobilized on a positioning tray and thenplaced on the Piximus imaging window. All scans were performed using thetotal body mode (0.18×0.18 mm), and the analysis was performed on thetotal body region of interest. The entire body, except the head, of eachmouse was exposed for 5 minutes to a cone shaped beam of both high andlow energy x-rays. A high-resolution digital picture was taken of theimage of the x-rays hitting a luminescent panel. Lunar PIXImus software(version 1.45) was used to calculate the ratio of attenuation of thehigh and low energies to separate bone from soft tissue compartmentsand, within the soft tissue compartment, to separate lean tissue massfrom fat mass and thus determine the bone mineral density (BMD),volumetric bone mineral density (vBMD), total bone mineral content(BMC), fat composition (% fat), lean body mass (LBM), the ratio ofBMC/LBM, and total tissue mass (TTM) in the regions of interest (totalbody for all tests, and additionally, vertebrae and both femurs forBMD). Previous studies have determined that this technique preciselymeasures fat and lean tissue mass, and that there is a closerelationship between fat and lean tissue mass estimated by thistechnique with those measured using chemical carcass analysis (Nagy andClair, 2000, Obesity Research 8:392-398).

[0216] Body composition and percent body fat was measured in eight (8)homozygous (6 males, 2 females), four (4) heterozygous (2 males and 2females), and four (4) wild-type (2 males and 2 females) mice. The meanpercent total body fat was notably decreased in both the male and female(−/−) mice compared to the (+/−) and (+/+) animals. However, the valuesfor the (−/−) animals fell within historical normal ranges. There was nodifference between groups in any of the other parameters measured (TTM,LBM, vBMD, total body BMD, femur BMD, vertebrae BMD, total body BMC, andthe BMC/LBM ratio).

[0217] Mouse body weight was determined to the nearest 0.1 gm using anOhaus Scout scale. Body length was determined from nose to the base oftail and is reported in cm. Body weight and body length data wereobtained for mice at eight (8) weeks of age. The body weight of eight(8) homozygous (6 males, 2 females), four (4) heterozygous (2 males and2 females), and four (4) wild-type (2 males and 2 females) mice wasdetermined and analyzed. Decreased body weight was noted for the twofemale (−/−) mice compared to the female (+/+) and (+/−) mice.

[0218] Body length data was determined and analyzed for eight (8)homozygous (6 males, 2 females), four (4) heterozygous (2 males and 2females), and four (4) wild-type (2 males and 2 females) mice. There wasno significant difference in body length between groups.

[0219] 6.1.3 The Effect of NHP Disruption on Hematology and BloodChemistry

[0220] Whole blood was collected by retro-orbital bleed and placed in acapillary blood collection tube that contained EDTA. The blood wasanalyzed using the Cell-Dyn 3500R analyzer (Abbott Diagnostics). Theanalyzer employs dual technologies to provide the basis for a five-partwhite blood cell (WBC) differential identification. Multi-AnglePolarized Scatter Separation (MAPSS) provides the primary white bloodcell count and differential information, while impedance providesadditional information in the presence of fragile lymphocytes andhypotonically resistant red blood cells. Approximately 135 microlitersof whole blood is aspirated into the analyzer using a peristaltic pump.Four independent measurement techniques are used by the Cell-Dyn 3500RSystem to obtain the hematologic parameters. The WBC Optical Count (WOC)and the WBC differential data are measured in the optical flow channel,resulting in the identification of the WBC subpopulations (neutrophils,lymphocytes, monocytes, eosinophils, and basophils) for the five partWBC differential. The WBC Impedance Count (WIC) is measured in oneelectrical impedance channel. The RBC and platelet data are measured ina second electrical impedance channel. The hemoglobin is measured in thespectrophotometric channel. The sample was aspirated, diluted, mixed,and the measurements for each parameter were obtained during eachinstrument cycle. The final hematological analysis parameters obtainedare white blood cell count, neutrophils, lymphocytes, monocytes,eosinophils, basophils, red blood cells, hemoglobin, hematocrit,platlets, red cell distribution width, mean corpuscular volume, meancorpuscular hemoglobin, mean corpuscular hemoglobin concentration andmean platelet volume.

[0221] Blood samples were obtained from a total of sixteen (16) mice;eight (8) homozygous (6 males, 2 females), four (4) heterozygous (2males and 2 females), and four (4) wild-type (2 males and 2 females)mice. Analysis and comparison of the blood samples revealed thatdisruption of the NHP gene results in an upward trend in mean whiteblood cell (WBC) counts, with subsequent increases in absoluteneutrophil (NEUTRO), lymphocyte (LYM), monocyte (MONO), eosinophil (EOS)and basophil (BASO) counts in the (−/−) and (+/−) mice. The WBC valuesfor the +/+ mice were 5.9±2.3, for the +/− mice were 9.7±0.7, and forthe −/− mice were 9.6±2.8. The NEUTRO values for the +/+ mice were0.91±0.29, for the +/− mice were 1.40±0.39, and for the −/− mice were1.26±0.34. The LYM values for the +/+ mice were 4.61±2.03, for the +/−mice were 7.70±0.53, and for the −/− mice were 7.83±2.58. The MONOvalues for the +/+ mice were 0.30±0.13, for the +/− mice were 0.53±0.19,and for the −/− mice were 0.42±0.18. The EOS values for the +/+ micewere 0.033±0.030, for the +/− mice were 0.063±0.050, and for the −/−mice were 0.050±0.059. The BASO values for the +/+ mice were0.008±0.008, for the +/− mice were 0.016±0.009, and for the −/− micewere 0.018±0.010. However, the relative distribution of these cellsremained normal.

[0222] Approximately 200 microliters of whole blood was collected fromthe retro-orbital plexus. The blood was placed in a 2.5 mlmicro-collection tube and centrifuged to obtain the serum. The samplewas analyzed for the following analytes: albumin, alkaline phosphatase,alanine aminotransferase (ALT), total bilirubin, blood urea nitrogen(BUN), calcium, glucose, phosphorus, cholesterol, triglycerides,creatinine and uric acid using a Cobas Integra 400 (Roche Diagnostics).The Cobas Integra 400 is a random and continuous access, sampleselective analyzer. The analyzer uses four measuring principles:absorbance photometry, turbidimetry, fluorescence polarimetry andion-selective electrode potentiometry to assay the analytes describedabove.

[0223] A total of sixteen (16) mice were analyzed; eight (8) homozygous(6 males, 2 females), four (4) heterozygous (2 males and 2 females), andfour (4) wild-type (2 males and 2 females) mice. There were nosignificant differences in any of the above analytes between the threegroups.

[0224] The mononuclear cell profile is derived by staining a singlesample of lysed peripheral blood from each mouse with a panel of sixlineage-specific antibodies: CD45 PerCP, anti-TCRb APC, CD4 PE, CD8FITC, pan-NK PE, and CD19 FITC. The two FITC and PE labeled antibodiesstain-mutually exclusive cell types. The samples are analyzed using aBecton Dickinson FACSCalibur flow cytometer with CellQuest software. Allleukocytes are identified by CD45 staining, and granulocytes areexcluded by scatter. T cells are identified by expression of TCRb-chain, and are further divided into CD4+CD8− (mature helper) andCD4−CD8+ (mature cytotoxic/suppressor). NK cells and B cells areidentified from the TCRb− (non-T) population by staining with pan-NK andCD19 antibody, respectively. Monocytes are defined as CD45+ mononuclearcells which are negative for all T, B, and NK markers.

[0225] A total of six (6) mice were analyzed: two (2) wild-type (+/+);two (2) heterozygotes (+/−); and two (2) homozygotes. There were nosignificant differences in the percentage of CD4+CD8− or CD4−CD8+ cells,the CD4+/CD8+ ratio, or the percentage of B cells or monocytes betweenthe two groups.

[0226] Additionally, quantitative insulin assays were performed on ofeight (8) homozygous mice (6 males and 2 females) and four (4) wild-typemice (2 males and 2 females) using a Cobra II Series Auto-Gamma CountingSystem to determine the insulin levels. There was no significantdifference in the insulin levels between the two groups of mice.

[0227] 6.1.4 Urinalysis

[0228] Approximately 100 microliters of urine was collected by placingthe mouse in a clean cage lined with aluminum foil and carefullywatching the mouse for urination. Immediately following urination, thesample was collected into a micro-collection tube. The specimen wasanalyzed using a calibrated Chemstrip 101 Urine Analyzer (AmesDiagnostics) urinalysis test strip. The urine was placed on the test padand was read as indicated by the manufacturer according to the packageinsert. This urinalysis determines urine osmolality, the presence ofleukocytes, nitrite, protein, glucose, ketones, urobilinogen, bilirubinand blood.

[0229] Urine samples were obtained from fifteen (15) mice; eight (8)homozygous (6 males, 2 females), four (4) heterozygous (2 males and 2females), and three (4) wild-type (2 males and 1 female) mice. Analysisand comparison of the urine samples revealed abnormal protein levels (30mg/dl) in 5 of the 8 homozygous mice (compared to only 1 of the 4heterozygous and 1 of the 3 wild-type mice), and abnormal ketone levelsin 6 of the 8 homozygous mice (compared to only 1 of the 4 heterozygousand 1 of the 3 wild-type mice). No differences between the groups in theother analytes were detected.

[0230] 6.1.5 Opthalmology

[0231] Slit Lamp Analysis: The slit lamp is a biomicroscope that allowsexamination of the anatomy of the anterior eye segment, as well as thelocalization of some abnormalities. It is a rapid and convenient methodfor preliminary eye examination prior to fundus photography. Mouse eyeanalysis began with examination utilizing a slit lamp (Nikon, Tokyo,Japan) in combination with a 60 or 90 diopter (D) condensing lens. Inpreparation for examination, mouse pupils were dilated by adding a dropof 1% cyclopentolate and 1% atropine (Alcon Laboratory Inc., Fort Worth,Tex.) to each eye.

[0232] Fundus Photography: Fundus photography is a noninvasive method ofexamining the eye that is adaptable to high throughput analysis. Theappearance of the ocular fundus is representative of overall health.Variation in the appearance of the ocular fundus can be indicative ofdifferent diseases, including, but not limited to, diabetes, obesity,cardiovascular disorders, angiogenesis, oxidant related disorders andcancer. Selected animals were subjected to fundus photography using aKowa Genesis small animal fundus digital camera (Tokyo, Japan) tophotograph mouse fundi. The instrument was used with a condensing lens,Volk 60D or 90D (Mentor, Ohio, USA), mounted between the camera and theobject to be viewed (mouse eye). In order to avoid complications ofanesthesia, such as clouding of the ocular media, photographs wereobtained on conscious mice, whose vibrissae were trimmed with finescissors to prevent them from obscuring the photograph.

[0233] Retinal Angiography: Fluor escein angiography is an establishedtechnique used to examine the circulation of the retina. In particularit enables the progression of diabetic retinopathy to be monitored, andprovides valuable information on the presence or absence of vascularlesions such as edema (leakage) and ischemia (occlusion of thecapillaries). The retinal angiography procedure was similar to theprocedure used for fundus photography, except that the standard lightwas replaced with blue light in combination with a barrier filter. Tofacilitate imaging, mice were injected intraperitoneally with 25% sodiumfluorescein (Akorn Inc., Decator, Ill.) at a dose of 0.01 ml per 5-6 gmbody weight. For viewing, the eyepiece was fitted with themanufacturer-supplied barrier filter. The digital imaging system usedconsists of a camera, a computer, and Komit+software (Kowa, Tokyo,Japan) especially designed for ophthalmological applications, whichfacilitates image data acquisition, analysis and storage.

[0234] The artery to vein ratio (A/V) is the ratio of the arterydiameter to the vein diameter (measured before the bifurcation of thevessels). The A/V ratio is measured and calculated according to fundusimages. Many diseases will influence the ratio, i.e., diabetes,cardiovascular disorders, papilledema and optic atrophy.

[0235] Ophthalmological analysis was performed on sixteen (16) mice;eight (8) homozygous (6 males, 2 females), four (4) heterozygous (2males and 2 females), and four (4) wild-type (2 males and 2 females).The analysis revealed no significant ophthalmological differencesbetween mice with or without functional NHP alleles, with the exceptionof an increased retinal arteriolar light reflex (ALR) noted in 4/8 (−/−)mice compared to the (+/+) mice, indicating abnormal thickening of thearteriolar wall. Additionally, ⅛ (−/−) mice had a subcapsular cataract.

[0236] 6.1.6 Neurological and Behavioral Analysis

[0237] Functional Observational Battery (FOB): A subset of tests fromthe Irwin neurological screen (Irwin, 1968, Psychopharmacologia13:222-257) were used to evaluate the gross neurological function of themice. This battery of simple neurological tests took 10 minutes and wasuseful for detecting severe neurological impairments.

[0238] Mice were first examined for their overall physicalcharacteristics, such as presence of whiskers, bald patches,piloerection, exopthalmus, palpebral closure, kyphosis, lordosis, andtail abnormalities. The mice were then placed into an empty cage for oneminute. Any abnormal spontaneous behaviors such as wild-running,excessive grooming, freezing behavior, hunched body posture whenwalking, etc., were recorded. Mice were next placed into an empty cageto assess motor reflexes. The cage was quickly moved side to side and upand down. The normal postural reflex is to extend all four legs in orderto maintain an upright, balanced position. The righting reflex wasmeasured by turning the mice on their back and determining how long ittook the mice to return to an upright position. Normal mice willimmediately right themselves. If a mouse did not right itself within 60seconds, the mouse was returned to its normal upright position.

[0239] The eye blink reflex, ear twitch reflex, and flank reflex weremeasured by lightly touching the eye, tip of the ear, and torso onceeach with a thin clear piece of plastic. The whisker-orienting responsewas measured by lightly touching the whiskers with a thin clear piece ofplastic while the animal was allowed to move freely. The whiskers aretypically moving. When touched the whiskers of normal mice will stopmoving and in many cases the mouse will turn its head to the side wherethe whiskers were touched. To determine a mouse's visual response tolight, the mouse was examined in a dimly lit room. Pupil constrictionand dilation reflexes were measured by quickly directing a penlight atthe mouse's eye and observing pupil constriction and subsequently pupildilatation once the light source was removed.

[0240] The visual reaching response was measured by suspending a mouseby its tail and moving it down towards the edge of a cage. A mouse thatcan see the cage will reach towards it when the cage is moved in thedirection of the mouse.

[0241] The tail suspension response was determined by holding the mouseapproximately six inches in the air by the tail for 20 seconds andrecording normal behaviors such as grabbing of the hindlimbs with theforelimbs and turning up on its sides. If present, abnormal behaviorssuch as hindlimb and forelimb clutch were also recorded.

[0242] The cateleptic response was measured by using a small rod at afixed vertical position. The mouse was positioned such that itsforelimbs were resting on the rod. Normal mice in this situation willquickly remove their forelimbs from the rod. A 60 second time limit wasallowed, after which a non-responsive mouse was returned to its normalposture.

[0243] The olfactory response was tested by using an odor such asvanilla extract as an olfactory cue. A small amount of vanilla wasplaced on cotton swab and held behind and to the side of a mouse. If themouse turns and orients itself to the position of the vanillaextract-containing cotton swab, the result is interpreted as anindication that the mouse can smell this olfactory cue.

[0244] Mouse body temperature was determined by gently inserting a smallprobe into the rectum and recording the temperature with a digitalread-out (Physitemp, Clifton). This process took less than 5 sec and themice appeared calm and unstressed throughout the procedure.

[0245] This entire battery of simple neurological tests took about 10minutes and provided for the detection of severe neurologicalimpairment. At the completion of these tests the mice were returned totheir home cage.

[0246] Hot Plate Assay for Nociception: Mice were removed from theirhome cage and placed on a 55.0° C. (+/−0.2° C.) hot plate, and thelatency to the first hind limb response was recorded. A Plexiglasenclosure was placed around the subject to keep them from walking off ofthe plate. The hind paw response is a foot shake, paw lick, or jump. Themaximum time allowed for a hind limb response to occur was 30 seconds,after which the mouse was removed if a hind limb response had notoccurred.

[0247] Open Field Assay for Anxiety Related Responses andLocomotor/Exploratory Activity: Anxiety-related, locomotor, andexploratory responses were measured in a clear Plexiglas (40 cm×40 cm×30cm) open-field arena. A mouse was placed in the center of the arena andallowed to explore for 20 minutes. Overhead high-level lighting providesadditional room lighting to enhance anxiety-related behaviors. Activityin the open field was quantified by a computer-controlled Vetsamaxoptical animal activity system (Accuscan Instruments, Columbus, Ohio)containing 16 photoreceptor beams on each side of the arena, therebydividing the arena into 256 equally-sized squares. An additional set ofphotobeams was placed above this set to record vertical activity, and aset was placed below to record nose poke activity, thus giving threelevels of recordable activity. Total distance traveled (locomotoractivity), number of rearing and nose poke events (exploratoryactivity), and center distance (i.e., the distance traveled in thecenter of the arena) were recorded. The center distance was divided bythe total distance traveled to obtain a center distance:total distanceratio. The center distance:total distance ratio can be used as an indexof anxiety-related responses. Data was collected in four-minuteintervals over the 20 minute test session.

[0248] Rotarod Assay for Motor Coordination: Motor coordination andbalance were tested using an accelerating rotarod (Accuscan Instruments,Columbus, Ohio). The test was performed by placing a mouse on a rotatingdrum (measuring 3 cm in diameter) and recording the time each animal wasable to stay on the rotating rod. Some mice hold on to the rotating rodas they begin to fall and ride completely around the rod. The speed ofthe rod accelerates from 0 to 40 rpm over the length of the 5 minutetest. Mice were given four consecutive trials with a maximum time of 300seconds (5 min).

[0249] Prepulse Inhibition of the Acoustic Startle Response: Prepulseinhibition of the acoustic startle response was measured using theSR-Lab System (San Diego Instruments, San Diego, Calif.). A test sessionbegan by placing a mouse in the Plexiglas cylinder where it was leftundisturbed for 3 min. A test session consists of three trial types. Onetrial type was a 40 ms, 120 decibel (dB) sound burst alone that istermed the startle stimulus. There were four different acoustic prepulseplus startle stimulus trial types. The prepulse sound is presented 100ms before the startle stimulus. The 20 ms prepulse sounds are at 74, 78,82, and 90 dB. Finally, there were trials where no stimulus is presentedto measure baseline movement in the cylinders. Six blocks of the sixtrial types were presented in pseudorandom order, such that each trialtype was presented once within a block of seven trials. The averageintertrial interval was 15 sec with a range of 10 to 20 seconds. Thestartle response is recorded for 65 ms (measuring the response every 1ms) starting at the onset of the startle stimulus. The background noiselevel in each chamber is approximately 70 dB. The maximum startleamplitude recorded during the 65 ms sampling window (Vmax) was used.

[0250] The formula used to calculate % prepulse inhibition of a startleresponse is:

[0251] 100-[(startle on acoustic prepulse+startle stimulustrials/startle response alone trials) ×100].

[0252] Sixteen (16) mice were analyzed; eight (8) homozygous (6 males, 2females), four (4) heterozygous (2 males and 2 females), and four (4)wild-type (2 males and 2 females) mice. There were no notabledifferences in any of the parameters measured between the groups.

[0253] 6.1.7 Radiology

[0254] One (1) male wild type (+/+) mouse and four (4) homozygous (−/−)mice (2 males and 2 females) were subject to examination using amouse-size computer aided tomography (CT) scanning unit, the MicroCAT™(ImTek, Inc., Knoxville, Tenn.). The mice were injected with a CTcontrast agent, omnipaque 300 (Nycomed Amersham, 300 mg of iodine perml, 0.25 ml per animal, or 2.50-3.75 g iodine/kg body weight),intraperitoneally. After resting in the cage for approximately 10minutes, the mice were sedated by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight). The CT-scan wasdone with the anesthetized animal lying prone on the test bed. Threedimensional images were reconstructed by the Feldkamp algorithm in acluster of workstations using ImTek 3DRECON software.

[0255] Significant depletion of fat depots in the abdominal andsubcutaneous regions was noted for the 2 female (−/−) mice. There wereno additional radiologic findings of significance that differentiatedthe mice of any genotypic group. The following observations were madefor all groups of mice. There were no abnormalities observed in theskull, spine, tail or individual bones. The head, brain and neckappeared normal. The cervical lymph nodes were not enlarged. The lungfields were clear. The hearts were of normal size. The mediastinum andvessels revealed no abnormalities. The liver, spleen and kidneys werenormal in size, shape and position. The rate of excretion of contrastmedia from the kidneys was within normal limits, indicating normalkidney function. The lymph nodes, and other abdominal organs, such asthe adrenals, ovaries and prostate were normal. No lesions were observedin the soft tissues (skin, muscle or fat).

[0256] 6.1.8 Blood Pressure and Heart Rate Determination

[0257] In an additional study, blood pressure was determined in eight(8) homozygous (6 males, 2 females) and four (4) wild-type (2 males and2 females) mice. Mice were subjected to a conscious systolic bloodpressure protocol similar to that previously described (Krege et al.,1995, Hypertension 25:1111-1115). Briefly, mice were placed on a heatedplatform (37° C.) with their tails placed through a cuff and in a sensorto detect the systolic blood pressure. The blood pressure was measured20 times a day for 4 consecutive days—the first ten measurements arediscarded to allow the animals to acclimate, and then the next tenmeasurements are recorded. There was no significant difference in theaverage systolic blood pressure between the wild-type mice and thehomozygous mice.

[0258] Additionally, the heart rate was measured in eight (8) homozygous(6 males, 2 females) and four (4) wild-type (2 males and 2 females)mice. There was no significant difference in the heart rate between thewild-type mice and the homozygous mice.

[0259] The present invention is not to be limited in scope by thespecific embodiments described herein, which are intended as singleillustrations of individual aspects of the invention, and functionallyequivalent methods and components are within the scope of the invention.Indeed, various modifications of the invention, in addition to thoseshown and described herein will become apparent to those skilled in theart from the foregoing description. Such modifications are intended tofall within the scope of the appended claims. All cited publications,patents, and patent applications are herein incorported by reference intheir entirety.

1 9 1 2220 DNA homo sapiens 1 atgctgtact ccccagggcc gagtcttccggagtcagcag agagcctgga tggatcacag 60 gaggataagc ctcggggctc atgtgcggagcccactttta ctgatacggg aatggtggct 120 cacataaaca acagccggct caaggccaagggcgtgggcc agcacgacaa cgcccagaac 180 tttggtaacc agagctttga ggagctgcgagcagcctgtc taagaaaggg ggagctcttc 240 gaggacccct tattccctgc tgaacccagctcactgggct tcaaggacct gggccccaac 300 tccaaaaatg tgcagaacat ctcctggcagcggcccaagg atatcataaa caaccctcta 360 ttcatcatgg atgggatttc tccaacagacatctgccagg ggatcctcgg ggactgctgg 420 ctgctggctg ccatcggctc ccttaccacctgccccaaac tgctataccg cgtggtgccc 480 agaggacaga gcttcaagaa aaactatgctggcatcttcc attttcagat ttggcagttt 540 ggacagtggg tgaacgtggt ggtagatgaccggctgccca caaagaatga caagctggtg 600 tttgtgcact caaccgaacg cagtgagttctggagtgccc tgctggagaa ggcgtatgcc 660 aagctgagtg ggtcctatga agcattgtcagggggcagta ccatggaggg ccttgaggac 720 ttcacaggag gcgtggccca gagcttccaactccagaggc cccctcagaa cctgctcagg 780 ctccttagga aggccgtgga gcgatcctccctcatgggtt gctccattga agtcaccagt 840 gatagtgaac tggaatccat gactgacaagatgctggtga gagggcacgc ttactctgtg 900 actggccttc aggatgtcca ctacagaggcaaaatggaaa cactgattcg ggtccggaat 960 ccctggggcc ggattgagtg gaatggagcttggagtgaca gtgccaggga gtgggaagag 1020 gtggcctcag acatccagat gcagctgctgcacaagacgg aggacgggga gttctggatg 1080 tcctaccaag atttcctgaa caacttcacgctcctggaga tctgcaacct cacgcctgat 1140 acactctctg gggactacaa gagctactggcacaccacct tctacgaggg cagctggcgc 1200 agaggcagct ccgcaggggg ctgcaggaaccaccctggca cgttctggac caacccccag 1260 tttaagatct ctcttcctga gggggatgacccagaggatg acgcagaggg caatgttgtg 1320 gtctgcacct gcctggtggc cctaatgcagaagaactggc ggcatgcacg gcagcaggga 1380 gcccagctgc agaccattgg ctttgtcctctacgcggtcc caaaagagtt tcagaacatt 1440 caggatgtcc acttgaagaa ggaattcttcacgaagtatc aggaccacgg cttctcagag 1500 atcttcacca actcacggga ggtgagcagccaactccggc tgcctccggg ggaatatatc 1560 attattccct ccacctttga gccacacagagatgctgact tcctgcttcg ggtcttcacc 1620 gagaagcaca gcgagtcatg ggaattggatgaagtcaact atgctgagca actccaagag 1680 gaaaaggtct ctgaggatga catggaccaggacttcctac atttgtttaa gatagtggca 1740 ggagagggca aggagatagg ggtgtatgagctccagaggc tgctcaacag gatggccatc 1800 aaattcaaaa gcttcaagac caagggctttggcctggatg cttgccgctg catgatcaac 1860 ctcatggata aagatggctc tggcaagctggggcttctag agttcaagat cctgtggaaa 1920 aaactcaaga aatggatgga catcttcagagagtgtgacc aggaccattc aggcaccttg 1980 aactcctatg agatgcgcct ggttattgagaaagcaggca tcaagctgaa caacaaggta 2040 atgcaggtcc tggtggccag gtatgcagatgatgacctga tcatagactt tgacagcttc 2100 atcagctgtt tcctgaggct aaagaccatgttcacattct ttctaaccat ggaccccaag 2160 aatactggcc atatttgctt gagcctggaacagtggctgc agatgaccat gtggggatag 2220 2 739 PRT homo sapiens 2 Met LeuTyr Ser Pro Gly Pro Ser Leu Pro Glu Ser Ala Glu Ser Leu 1 5 10 15 AspGly Ser Gln Glu Asp Lys Pro Arg Gly Ser Cys Ala Glu Pro Thr 20 25 30 PheThr Asp Thr Gly Met Val Ala His Ile Asn Asn Ser Arg Leu Lys 35 40 45 AlaLys Gly Val Gly Gln His Asp Asn Ala Gln Asn Phe Gly Asn Gln 50 55 60 SerPhe Glu Glu Leu Arg Ala Ala Cys Leu Arg Lys Gly Glu Leu Phe 65 70 75 80Glu Asp Pro Leu Phe Pro Ala Glu Pro Ser Ser Leu Gly Phe Lys Asp 85 90 95Leu Gly Pro Asn Ser Lys Asn Val Gln Asn Ile Ser Trp Gln Arg Pro 100 105110 Lys Asp Ile Ile Asn Asn Pro Leu Phe Ile Met Asp Gly Ile Ser Pro 115120 125 Thr Asp Ile Cys Gln Gly Ile Leu Gly Asp Cys Trp Leu Leu Ala Ala130 135 140 Ile Gly Ser Leu Thr Thr Cys Pro Lys Leu Leu Tyr Arg Val ValPro 145 150 155 160 Arg Gly Gln Ser Phe Lys Lys Asn Tyr Ala Gly Ile PheHis Phe Gln 165 170 175 Ile Trp Gln Phe Gly Gln Trp Val Asn Val Val ValAsp Asp Arg Leu 180 185 190 Pro Thr Lys Asn Asp Lys Leu Val Phe Val HisSer Thr Glu Arg Ser 195 200 205 Glu Phe Trp Ser Ala Leu Leu Glu Lys AlaTyr Ala Lys Leu Ser Gly 210 215 220 Ser Tyr Glu Ala Leu Ser Gly Gly SerThr Met Glu Gly Leu Glu Asp 225 230 235 240 Phe Thr Gly Gly Val Ala GlnSer Phe Gln Leu Gln Arg Pro Pro Gln 245 250 255 Asn Leu Leu Arg Leu LeuArg Lys Ala Val Glu Arg Ser Ser Leu Met 260 265 270 Gly Cys Ser Ile GluVal Thr Ser Asp Ser Glu Leu Glu Ser Met Thr 275 280 285 Asp Lys Met LeuVal Arg Gly His Ala Tyr Ser Val Thr Gly Leu Gln 290 295 300 Asp Val HisTyr Arg Gly Lys Met Glu Thr Leu Ile Arg Val Arg Asn 305 310 315 320 ProTrp Gly Arg Ile Glu Trp Asn Gly Ala Trp Ser Asp Ser Ala Arg 325 330 335Glu Trp Glu Glu Val Ala Ser Asp Ile Gln Met Gln Leu Leu His Lys 340 345350 Thr Glu Asp Gly Glu Phe Trp Met Ser Tyr Gln Asp Phe Leu Asn Asn 355360 365 Phe Thr Leu Leu Glu Ile Cys Asn Leu Thr Pro Asp Thr Leu Ser Gly370 375 380 Asp Tyr Lys Ser Tyr Trp His Thr Thr Phe Tyr Glu Gly Ser TrpArg 385 390 395 400 Arg Gly Ser Ser Ala Gly Gly Cys Arg Asn His Pro GlyThr Phe Trp 405 410 415 Thr Asn Pro Gln Phe Lys Ile Ser Leu Pro Glu GlyAsp Asp Pro Glu 420 425 430 Asp Asp Ala Glu Gly Asn Val Val Val Cys ThrCys Leu Val Ala Leu 435 440 445 Met Gln Lys Asn Trp Arg His Ala Arg GlnGln Gly Ala Gln Leu Gln 450 455 460 Thr Ile Gly Phe Val Leu Tyr Ala ValPro Lys Glu Phe Gln Asn Ile 465 470 475 480 Gln Asp Val His Leu Lys LysGlu Phe Phe Thr Lys Tyr Gln Asp His 485 490 495 Gly Phe Ser Glu Ile PheThr Asn Ser Arg Glu Val Ser Ser Gln Leu 500 505 510 Arg Leu Pro Pro GlyGlu Tyr Ile Ile Ile Pro Ser Thr Phe Glu Pro 515 520 525 His Arg Asp AlaAsp Phe Leu Leu Arg Val Phe Thr Glu Lys His Ser 530 535 540 Glu Ser TrpGlu Leu Asp Glu Val Asn Tyr Ala Glu Gln Leu Gln Glu 545 550 555 560 GluLys Val Ser Glu Asp Asp Met Asp Gln Asp Phe Leu His Leu Phe 565 570 575Lys Ile Val Ala Gly Glu Gly Lys Glu Ile Gly Val Tyr Glu Leu Gln 580 585590 Arg Leu Leu Asn Arg Met Ala Ile Lys Phe Lys Ser Phe Lys Thr Lys 595600 605 Gly Phe Gly Leu Asp Ala Cys Arg Cys Met Ile Asn Leu Met Asp Lys610 615 620 Asp Gly Ser Gly Lys Leu Gly Leu Leu Glu Phe Lys Ile Leu TrpLys 625 630 635 640 Lys Leu Lys Lys Trp Met Asp Ile Phe Arg Glu Cys AspGln Asp His 645 650 655 Ser Gly Thr Leu Asn Ser Tyr Glu Met Arg Leu ValIle Glu Lys Ala 660 665 670 Gly Ile Lys Leu Asn Asn Lys Val Met Gln ValLeu Val Ala Arg Tyr 675 680 685 Ala Asp Asp Asp Leu Ile Ile Asp Phe AspSer Phe Ile Ser Cys Phe 690 695 700 Leu Arg Leu Lys Thr Met Phe Thr PhePhe Leu Thr Met Asp Pro Lys 705 710 715 720 Asn Thr Gly His Ile Cys LeuSer Leu Glu Gln Trp Leu Gln Met Thr 725 730 735 Met Trp Gly 3 2172 DNAhomo sapiens 3 atgctgtact ccccagggcc gagtcttccg gagtcagcag agagcctggatggatcacag 60 gaggataagc ctcggggctc atgtgcggag cccactttta ctgatacgggaatggtggct 120 cacataaaca acagccggct caaggccaag ggcgtgggcc agcacgacaacgcccagaac 180 tttggtaacc agagctttga ggagctgcga gcagcctgtc taagaaagggggagctcttc 240 gaggacccct tattccctgc tgaacccagc tcactgggct tcaaggacctgggccccaac 300 tccaaaaatg tgcagaacat ctcctggcag cggcccaagg atatcataaacaaccctcta 360 ttcatcatgg atgggatttc tccaacagac atctgccagg ggatcctcggggactgctgg 420 ctgctggctg ccatcggctc ccttaccacc tgccccaaac tgctataccgcgtggtgccc 480 agaggacaga gcttcaagaa aaactatgct ggcatcttcc attttcagatttggcagttt 540 ggacagtggg tgaacgtggt ggtagatgac cggctgccca caaagaatgacaagctggtg 600 tttgtgcact caaccgaacg cagtgagttc tggagtgccc tgctggagaaggcgtatgcc 660 aagctgagtg ggtcctatga agcattgtca gggggcagta ccatggagggccttgaggac 720 ttcacaggag gcgtggccca gagcttccaa ctccagaggc cccctcagaacctgctcagg 780 ctccttagga aggccgtgga gcgatcctcc ctcatgggtt gctccattgaagtcaccagt 840 gatagtgaac tggaatccat gactgacaag atgctggtga gagggcacgcttactctgtg 900 actggccttc aggatgtcca ctacagaggc aaaatggaaa cactgattcgggtccggaat 960 ccctggggcc ggattgagtg gaatggagct tggagtgaca gtgccagggagtgggaagag 1020 gtggcctcag acatccagat gcagctgctg cacaagacgg aggacggggagttctggatg 1080 tcctaccaag atttcctgaa caacttcacg ctcctggaga tctgcaacctcacgcctgat 1140 acactctctg gggactacaa gagctactgg cacaccacct tctacgagggcagctggcgc 1200 agaggcagct ccgcaggggg ctgcaggaac caccctggca cgttctggaccaacccccag 1260 tttaagatct ctcttcctga gggggatgac ccagaggatg acgcagagggcaatgttgtg 1320 gtctgcacct gcctggtggc cctaatgcag aagaactggc ggcatgcacggcagcaggga 1380 gcccagctgc agaccattgg ctttgtcctc tacgcggtcc caaaagagtttcagaacatt 1440 caggatgtcc acttgaagaa ggaattcttc acgaagtatc aggaccacggcttctcagag 1500 atcttcacca actcacggga ggtgagcagc caactccggc tgcctccgggggaatatatc 1560 attattccct ccacctttga gccacacaga gatgctgact tcctgcttcgggtcttcacc 1620 gagaagcaca gcgagtcatg ggaattggat gaagtcaact atgctgagcaactccaagag 1680 gaaaaggtct ctgaggatga catggaccag gacttcctac atttgtttaagatagtggca 1740 ggagagggca aggagatagg ggtgtatgag ctccagaggc tgctcaacaggatggccatc 1800 aaattcaaaa gcttcaagac caagggcttt ggcctggatg cttgccgctgcatgatcaac 1860 ctcatggata aagatggctc tggcaagctg gggcttctag agttcaagatcctgtggaaa 1920 aaactcaaga aatggatgga catcttcaga gagtgtgacc aggaccattcaggcaccttg 1980 aactcctatg agatgcgcct ggttattgag aaagcaggca tcaagctgaacaacaaggta 2040 atgcaggtcc tggtggccag gtatgcagat gatgacctga tcatagactttgacagcttc 2100 atcagctgtt tcctgaggct aaagaccatg ttcatggctg cagatgaccatgtggggata 2160 gaggcgctgt ag 2172 4 723 PRT homo sapiens 4 Met Leu TyrSer Pro Gly Pro Ser Leu Pro Glu Ser Ala Glu Ser Leu 1 5 10 15 Asp GlySer Gln Glu Asp Lys Pro Arg Gly Ser Cys Ala Glu Pro Thr 20 25 30 Phe ThrAsp Thr Gly Met Val Ala His Ile Asn Asn Ser Arg Leu Lys 35 40 45 Ala LysGly Val Gly Gln His Asp Asn Ala Gln Asn Phe Gly Asn Gln 50 55 60 Ser PheGlu Glu Leu Arg Ala Ala Cys Leu Arg Lys Gly Glu Leu Phe 65 70 75 80 GluAsp Pro Leu Phe Pro Ala Glu Pro Ser Ser Leu Gly Phe Lys Asp 85 90 95 LeuGly Pro Asn Ser Lys Asn Val Gln Asn Ile Ser Trp Gln Arg Pro 100 105 110Lys Asp Ile Ile Asn Asn Pro Leu Phe Ile Met Asp Gly Ile Ser Pro 115 120125 Thr Asp Ile Cys Gln Gly Ile Leu Gly Asp Cys Trp Leu Leu Ala Ala 130135 140 Ile Gly Ser Leu Thr Thr Cys Pro Lys Leu Leu Tyr Arg Val Val Pro145 150 155 160 Arg Gly Gln Ser Phe Lys Lys Asn Tyr Ala Gly Ile Phe HisPhe Gln 165 170 175 Ile Trp Gln Phe Gly Gln Trp Val Asn Val Val Val AspAsp Arg Leu 180 185 190 Pro Thr Lys Asn Asp Lys Leu Val Phe Val His SerThr Glu Arg Ser 195 200 205 Glu Phe Trp Ser Ala Leu Leu Glu Lys Ala TyrAla Lys Leu Ser Gly 210 215 220 Ser Tyr Glu Ala Leu Ser Gly Gly Ser ThrMet Glu Gly Leu Glu Asp 225 230 235 240 Phe Thr Gly Gly Val Ala Gln SerPhe Gln Leu Gln Arg Pro Pro Gln 245 250 255 Asn Leu Leu Arg Leu Leu ArgLys Ala Val Glu Arg Ser Ser Leu Met 260 265 270 Gly Cys Ser Ile Glu ValThr Ser Asp Ser Glu Leu Glu Ser Met Thr 275 280 285 Asp Lys Met Leu ValArg Gly His Ala Tyr Ser Val Thr Gly Leu Gln 290 295 300 Asp Val His TyrArg Gly Lys Met Glu Thr Leu Ile Arg Val Arg Asn 305 310 315 320 Pro TrpGly Arg Ile Glu Trp Asn Gly Ala Trp Ser Asp Ser Ala Arg 325 330 335 GluTrp Glu Glu Val Ala Ser Asp Ile Gln Met Gln Leu Leu His Lys 340 345 350Thr Glu Asp Gly Glu Phe Trp Met Ser Tyr Gln Asp Phe Leu Asn Asn 355 360365 Phe Thr Leu Leu Glu Ile Cys Asn Leu Thr Pro Asp Thr Leu Ser Gly 370375 380 Asp Tyr Lys Ser Tyr Trp His Thr Thr Phe Tyr Glu Gly Ser Trp Arg385 390 395 400 Arg Gly Ser Ser Ala Gly Gly Cys Arg Asn His Pro Gly ThrPhe Trp 405 410 415 Thr Asn Pro Gln Phe Lys Ile Ser Leu Pro Glu Gly AspAsp Pro Glu 420 425 430 Asp Asp Ala Glu Gly Asn Val Val Val Cys Thr CysLeu Val Ala Leu 435 440 445 Met Gln Lys Asn Trp Arg His Ala Arg Gln GlnGly Ala Gln Leu Gln 450 455 460 Thr Ile Gly Phe Val Leu Tyr Ala Val ProLys Glu Phe Gln Asn Ile 465 470 475 480 Gln Asp Val His Leu Lys Lys GluPhe Phe Thr Lys Tyr Gln Asp His 485 490 495 Gly Phe Ser Glu Ile Phe ThrAsn Ser Arg Glu Val Ser Ser Gln Leu 500 505 510 Arg Leu Pro Pro Gly GluTyr Ile Ile Ile Pro Ser Thr Phe Glu Pro 515 520 525 His Arg Asp Ala AspPhe Leu Leu Arg Val Phe Thr Glu Lys His Ser 530 535 540 Glu Ser Trp GluLeu Asp Glu Val Asn Tyr Ala Glu Gln Leu Gln Glu 545 550 555 560 Glu LysVal Ser Glu Asp Asp Met Asp Gln Asp Phe Leu His Leu Phe 565 570 575 LysIle Val Ala Gly Glu Gly Lys Glu Ile Gly Val Tyr Glu Leu Gln 580 585 590Arg Leu Leu Asn Arg Met Ala Ile Lys Phe Lys Ser Phe Lys Thr Lys 595 600605 Gly Phe Gly Leu Asp Ala Cys Arg Cys Met Ile Asn Leu Met Asp Lys 610615 620 Asp Gly Ser Gly Lys Leu Gly Leu Leu Glu Phe Lys Ile Leu Trp Lys625 630 635 640 Lys Leu Lys Lys Trp Met Asp Ile Phe Arg Glu Cys Asp GlnAsp His 645 650 655 Ser Gly Thr Leu Asn Ser Tyr Glu Met Arg Leu Val IleGlu Lys Ala 660 665 670 Gly Ile Lys Leu Asn Asn Lys Val Met Gln Val LeuVal Ala Arg Tyr 675 680 685 Ala Asp Asp Asp Leu Ile Ile Asp Phe Asp SerPhe Ile Ser Cys Phe 690 695 700 Leu Arg Leu Lys Thr Met Phe Met Ala AlaAsp Asp His Val Gly Ile 705 710 715 720 Glu Ala Leu 5 2109 DNA homosapiens 5 atggtggctc acataaacaa cagccggctc aaggccaagg gcgtgggccagcacgacaac 60 gcccagaact ttggtaacca gagctttgag gagctgcgag cagcctgtctaagaaagggg 120 gagctcttcg aggacccctt attccctgct gaacccagct cactgggcttcaaggacctg 180 ggccccaact ccaaaaatgt gcagaacatc tcctggcagc ggcccaaggatatcataaac 240 aaccctctat tcatcatgga tgggatttct ccaacagaca tctgccaggggatcctcggg 300 gactgctggc tgctggctgc catcggctcc cttaccacct gccccaaactgctataccgc 360 gtggtgccca gaggacagag cttcaagaaa aactatgctg gcatcttccattttcagatt 420 tggcagtttg gacagtgggt gaacgtggtg gtagatgacc ggctgcccacaaagaatgac 480 aagctggtgt ttgtgcactc aaccgaacgc agtgagttct ggagtgccctgctggagaag 540 gcgtatgcca agctgagtgg gtcctatgaa gcattgtcag ggggcagtaccatggagggc 600 cttgaggact tcacaggagg cgtggcccag agcttccaac tccagaggccccctcagaac 660 ctgctcaggc tccttaggaa ggccgtggag cgatcctccc tcatgggttgctccattgaa 720 gtcaccagtg atagtgaact ggaatccatg actgacaaga tgctggtgagagggcacgct 780 tactctgtga ctggccttca ggatgtccac tacagaggca aaatggaaacactgattcgg 840 gtccggaatc cctggggccg gattgagtgg aatggagctt ggagtgacagtgccagggag 900 tgggaagagg tggcctcaga catccagatg cagctgctgc acaagacggaggacggggag 960 ttctggatgt cctaccaaga tttcctgaac aacttcacgc tcctggagatctgcaacctc 1020 acgcctgata cactctctgg ggactacaag agctactggc acaccaccttctacgagggc 1080 agctggcgca gaggcagctc cgcagggggc tgcaggaacc accctggcacgttctggacc 1140 aacccccagt ttaagatctc tcttcctgag ggggatgacc cagaggatgacgcagagggc 1200 aatgttgtgg tctgcacctg cctggtggcc ctaatgcaga agaactggcggcatgcacgg 1260 cagcagggag cccagctgca gaccattggc tttgtcctct acgcggtcccaaaagagttt 1320 cagaacattc aggatgtcca cttgaagaag gaattcttca cgaagtatcaggaccacggc 1380 ttctcagaga tcttcaccaa ctcacgggag gtgagcagcc aactccggctgcctccgggg 1440 gaatatatca ttattccctc cacctttgag ccacacagag atgctgacttcctgcttcgg 1500 gtcttcaccg agaagcacag cgagtcatgg gaattggatg aagtcaactatgctgagcaa 1560 ctccaagagg aaaaggtctc tgaggatgac atggaccagg acttcctacatttgtttaag 1620 atagtggcag gagagggcaa ggagataggg gtgtatgagc tccagaggctgctcaacagg 1680 atggccatca aattcaaaag cttcaagacc aagggctttg gcctggatgcttgccgctgc 1740 atgatcaacc tcatggataa agatggctct ggcaagctgg ggcttctagagttcaagatc 1800 ctgtggaaaa aactcaagaa atggatggac atcttcagag agtgtgaccaggaccattca 1860 ggcaccttga actcctatga gatgcgcctg gttattgaga aagcaggcatcaagctgaac 1920 aacaaggtaa tgcaggtcct ggtggccagg tatgcagatg atgacctgatcatagacttt 1980 gacagcttca tcagctgttt cctgaggcta aagaccatgt tcacattctttctaaccatg 2040 gaccccaaga atactggcca tatttgcttg agcctggaac agtggctgcagatgaccatg 2100 tggggatag 2109 6 702 PRT homo sapiens 6 Met Val Ala HisIle Asn Asn Ser Arg Leu Lys Ala Lys Gly Val Gly 1 5 10 15 Gln His AspAsn Ala Gln Asn Phe Gly Asn Gln Ser Phe Glu Glu Leu 20 25 30 Arg Ala AlaCys Leu Arg Lys Gly Glu Leu Phe Glu Asp Pro Leu Phe 35 40 45 Pro Ala GluPro Ser Ser Leu Gly Phe Lys Asp Leu Gly Pro Asn Ser 50 55 60 Lys Asn ValGln Asn Ile Ser Trp Gln Arg Pro Lys Asp Ile Ile Asn 65 70 75 80 Asn ProLeu Phe Ile Met Asp Gly Ile Ser Pro Thr Asp Ile Cys Gln 85 90 95 Gly IleLeu Gly Asp Cys Trp Leu Leu Ala Ala Ile Gly Ser Leu Thr 100 105 110 ThrCys Pro Lys Leu Leu Tyr Arg Val Val Pro Arg Gly Gln Ser Phe 115 120 125Lys Lys Asn Tyr Ala Gly Ile Phe His Phe Gln Ile Trp Gln Phe Gly 130 135140 Gln Trp Val Asn Val Val Val Asp Asp Arg Leu Pro Thr Lys Asn Asp 145150 155 160 Lys Leu Val Phe Val His Ser Thr Glu Arg Ser Glu Phe Trp SerAla 165 170 175 Leu Leu Glu Lys Ala Tyr Ala Lys Leu Ser Gly Ser Tyr GluAla Leu 180 185 190 Ser Gly Gly Ser Thr Met Glu Gly Leu Glu Asp Phe ThrGly Gly Val 195 200 205 Ala Gln Ser Phe Gln Leu Gln Arg Pro Pro Gln AsnLeu Leu Arg Leu 210 215 220 Leu Arg Lys Ala Val Glu Arg Ser Ser Leu MetGly Cys Ser Ile Glu 225 230 235 240 Val Thr Ser Asp Ser Glu Leu Glu SerMet Thr Asp Lys Met Leu Val 245 250 255 Arg Gly His Ala Tyr Ser Val ThrGly Leu Gln Asp Val His Tyr Arg 260 265 270 Gly Lys Met Glu Thr Leu IleArg Val Arg Asn Pro Trp Gly Arg Ile 275 280 285 Glu Trp Asn Gly Ala TrpSer Asp Ser Ala Arg Glu Trp Glu Glu Val 290 295 300 Ala Ser Asp Ile GlnMet Gln Leu Leu His Lys Thr Glu Asp Gly Glu 305 310 315 320 Phe Trp MetSer Tyr Gln Asp Phe Leu Asn Asn Phe Thr Leu Leu Glu 325 330 335 Ile CysAsn Leu Thr Pro Asp Thr Leu Ser Gly Asp Tyr Lys Ser Tyr 340 345 350 TrpHis Thr Thr Phe Tyr Glu Gly Ser Trp Arg Arg Gly Ser Ser Ala 355 360 365Gly Gly Cys Arg Asn His Pro Gly Thr Phe Trp Thr Asn Pro Gln Phe 370 375380 Lys Ile Ser Leu Pro Glu Gly Asp Asp Pro Glu Asp Asp Ala Glu Gly 385390 395 400 Asn Val Val Val Cys Thr Cys Leu Val Ala Leu Met Gln Lys AsnTrp 405 410 415 Arg His Ala Arg Gln Gln Gly Ala Gln Leu Gln Thr Ile GlyPhe Val 420 425 430 Leu Tyr Ala Val Pro Lys Glu Phe Gln Asn Ile Gln AspVal His Leu 435 440 445 Lys Lys Glu Phe Phe Thr Lys Tyr Gln Asp His GlyPhe Ser Glu Ile 450 455 460 Phe Thr Asn Ser Arg Glu Val Ser Ser Gln LeuArg Leu Pro Pro Gly 465 470 475 480 Glu Tyr Ile Ile Ile Pro Ser Thr PheGlu Pro His Arg Asp Ala Asp 485 490 495 Phe Leu Leu Arg Val Phe Thr GluLys His Ser Glu Ser Trp Glu Leu 500 505 510 Asp Glu Val Asn Tyr Ala GluGln Leu Gln Glu Glu Lys Val Ser Glu 515 520 525 Asp Asp Met Asp Gln AspPhe Leu His Leu Phe Lys Ile Val Ala Gly 530 535 540 Glu Gly Lys Glu IleGly Val Tyr Glu Leu Gln Arg Leu Leu Asn Arg 545 550 555 560 Met Ala IleLys Phe Lys Ser Phe Lys Thr Lys Gly Phe Gly Leu Asp 565 570 575 Ala CysArg Cys Met Ile Asn Leu Met Asp Lys Asp Gly Ser Gly Lys 580 585 590 LeuGly Leu Leu Glu Phe Lys Ile Leu Trp Lys Lys Leu Lys Lys Trp 595 600 605Met Asp Ile Phe Arg Glu Cys Asp Gln Asp His Ser Gly Thr Leu Asn 610 615620 Ser Tyr Glu Met Arg Leu Val Ile Glu Lys Ala Gly Ile Lys Leu Asn 625630 635 640 Asn Lys Val Met Gln Val Leu Val Ala Arg Tyr Ala Asp Asp AspLeu 645 650 655 Ile Ile Asp Phe Asp Ser Phe Ile Ser Cys Phe Leu Arg LeuLys Thr 660 665 670 Met Phe Thr Phe Phe Leu Thr Met Asp Pro Lys Asn ThrGly His Ile 675 680 685 Cys Leu Ser Leu Glu Gln Trp Leu Gln Met Thr MetTrp Gly 690 695 700 7 2061 DNA homo sapiens 7 atggtggctc acataaacaacagccggctc aaggccaagg gcgtgggcca gcacgacaac 60 gcccagaact ttggtaaccagagctttgag gagctgcgag cagcctgtct aagaaagggg 120 gagctcttcg aggaccccttattccctgct gaacccagct cactgggctt caaggacctg 180 ggccccaact ccaaaaatgtgcagaacatc tcctggcagc ggcccaagga tatcataaac 240 aaccctctat tcatcatggatgggatttct ccaacagaca tctgccaggg gatcctcggg 300 gactgctggc tgctggctgccatcggctcc cttaccacct gccccaaact gctataccgc 360 gtggtgccca gaggacagagcttcaagaaa aactatgctg gcatcttcca ttttcagatt 420 tggcagtttg gacagtgggtgaacgtggtg gtagatgacc ggctgcccac aaagaatgac 480 aagctggtgt ttgtgcactcaaccgaacgc agtgagttct ggagtgccct gctggagaag 540 gcgtatgcca agctgagtgggtcctatgaa gcattgtcag ggggcagtac catggagggc 600 cttgaggact tcacaggaggcgtggcccag agcttccaac tccagaggcc ccctcagaac 660 ctgctcaggc tccttaggaaggccgtggag cgatcctccc tcatgggttg ctccattgaa 720 gtcaccagtg atagtgaactggaatccatg actgacaaga tgctggtgag agggcacgct 780 tactctgtga ctggccttcaggatgtccac tacagaggca aaatggaaac actgattcgg 840 gtccggaatc cctggggccggattgagtgg aatggagctt ggagtgacag tgccagggag 900 tgggaagagg tggcctcagacatccagatg cagctgctgc acaagacgga ggacggggag 960 ttctggatgt cctaccaagatttcctgaac aacttcacgc tcctggagat ctgcaacctc 1020 acgcctgata cactctctggggactacaag agctactggc acaccacctt ctacgagggc 1080 agctggcgca gaggcagctccgcagggggc tgcaggaacc accctggcac gttctggacc 1140 aacccccagt ttaagatctctcttcctgag ggggatgacc cagaggatga cgcagagggc 1200 aatgttgtgg tctgcacctgcctggtggcc ctaatgcaga agaactggcg gcatgcacgg 1260 cagcagggag cccagctgcagaccattggc tttgtcctct acgcggtccc aaaagagttt 1320 cagaacattc aggatgtccacttgaagaag gaattcttca cgaagtatca ggaccacggc 1380 ttctcagaga tcttcaccaactcacgggag gtgagcagcc aactccggct gcctccgggg 1440 gaatatatca ttattccctccacctttgag ccacacagag atgctgactt cctgcttcgg 1500 gtcttcaccg agaagcacagcgagtcatgg gaattggatg aagtcaacta tgctgagcaa 1560 ctccaagagg aaaaggtctctgaggatgac atggaccagg acttcctaca tttgtttaag 1620 atagtggcag gagagggcaaggagataggg gtgtatgagc tccagaggct gctcaacagg 1680 atggccatca aattcaaaagcttcaagacc aagggctttg gcctggatgc ttgccgctgc 1740 atgatcaacc tcatggataaagatggctct ggcaagctgg ggcttctaga gttcaagatc 1800 ctgtggaaaa aactcaagaaatggatggac atcttcagag agtgtgacca ggaccattca 1860 ggcaccttga actcctatgagatgcgcctg gttattgaga aagcaggcat caagctgaac 1920 aacaaggtaa tgcaggtcctggtggccagg tatgcagatg atgacctgat catagacttt 1980 gacagcttca tcagctgtttcctgaggcta aagaccatgt tcatggctgc agatgaccat 2040 gtggggatag aggcgctgta g2061 8 686 PRT homo sapiens 8 Met Val Ala His Ile Asn Asn Ser Arg LeuLys Ala Lys Gly Val Gly 1 5 10 15 Gln His Asp Asn Ala Gln Asn Phe GlyAsn Gln Ser Phe Glu Glu Leu 20 25 30 Arg Ala Ala Cys Leu Arg Lys Gly GluLeu Phe Glu Asp Pro Leu Phe 35 40 45 Pro Ala Glu Pro Ser Ser Leu Gly PheLys Asp Leu Gly Pro Asn Ser 50 55 60 Lys Asn Val Gln Asn Ile Ser Trp GlnArg Pro Lys Asp Ile Ile Asn 65 70 75 80 Asn Pro Leu Phe Ile Met Asp GlyIle Ser Pro Thr Asp Ile Cys Gln 85 90 95 Gly Ile Leu Gly Asp Cys Trp LeuLeu Ala Ala Ile Gly Ser Leu Thr 100 105 110 Thr Cys Pro Lys Leu Leu TyrArg Val Val Pro Arg Gly Gln Ser Phe 115 120 125 Lys Lys Asn Tyr Ala GlyIle Phe His Phe Gln Ile Trp Gln Phe Gly 130 135 140 Gln Trp Val Asn ValVal Val Asp Asp Arg Leu Pro Thr Lys Asn Asp 145 150 155 160 Lys Leu ValPhe Val His Ser Thr Glu Arg Ser Glu Phe Trp Ser Ala 165 170 175 Leu LeuGlu Lys Ala Tyr Ala Lys Leu Ser Gly Ser Tyr Glu Ala Leu 180 185 190 SerGly Gly Ser Thr Met Glu Gly Leu Glu Asp Phe Thr Gly Gly Val 195 200 205Ala Gln Ser Phe Gln Leu Gln Arg Pro Pro Gln Asn Leu Leu Arg Leu 210 215220 Leu Arg Lys Ala Val Glu Arg Ser Ser Leu Met Gly Cys Ser Ile Glu 225230 235 240 Val Thr Ser Asp Ser Glu Leu Glu Ser Met Thr Asp Lys Met LeuVal 245 250 255 Arg Gly His Ala Tyr Ser Val Thr Gly Leu Gln Asp Val HisTyr Arg 260 265 270 Gly Lys Met Glu Thr Leu Ile Arg Val Arg Asn Pro TrpGly Arg Ile 275 280 285 Glu Trp Asn Gly Ala Trp Ser Asp Ser Ala Arg GluTrp Glu Glu Val 290 295 300 Ala Ser Asp Ile Gln Met Gln Leu Leu His LysThr Glu Asp Gly Glu 305 310 315 320 Phe Trp Met Ser Tyr Gln Asp Phe LeuAsn Asn Phe Thr Leu Leu Glu 325 330 335 Ile Cys Asn Leu Thr Pro Asp ThrLeu Ser Gly Asp Tyr Lys Ser Tyr 340 345 350 Trp His Thr Thr Phe Tyr GluGly Ser Trp Arg Arg Gly Ser Ser Ala 355 360 365 Gly Gly Cys Arg Asn HisPro Gly Thr Phe Trp Thr Asn Pro Gln Phe 370 375 380 Lys Ile Ser Leu ProGlu Gly Asp Asp Pro Glu Asp Asp Ala Glu Gly 385 390 395 400 Asn Val ValVal Cys Thr Cys Leu Val Ala Leu Met Gln Lys Asn Trp 405 410 415 Arg HisAla Arg Gln Gln Gly Ala Gln Leu Gln Thr Ile Gly Phe Val 420 425 430 LeuTyr Ala Val Pro Lys Glu Phe Gln Asn Ile Gln Asp Val His Leu 435 440 445Lys Lys Glu Phe Phe Thr Lys Tyr Gln Asp His Gly Phe Ser Glu Ile 450 455460 Phe Thr Asn Ser Arg Glu Val Ser Ser Gln Leu Arg Leu Pro Pro Gly 465470 475 480 Glu Tyr Ile Ile Ile Pro Ser Thr Phe Glu Pro His Arg Asp AlaAsp 485 490 495 Phe Leu Leu Arg Val Phe Thr Glu Lys His Ser Glu Ser TrpGlu Leu 500 505 510 Asp Glu Val Asn Tyr Ala Glu Gln Leu Gln Glu Glu LysVal Ser Glu 515 520 525 Asp Asp Met Asp Gln Asp Phe Leu His Leu Phe LysIle Val Ala Gly 530 535 540 Glu Gly Lys Glu Ile Gly Val Tyr Glu Leu GlnArg Leu Leu Asn Arg 545 550 555 560 Met Ala Ile Lys Phe Lys Ser Phe LysThr Lys Gly Phe Gly Leu Asp 565 570 575 Ala Cys Arg Cys Met Ile Asn LeuMet Asp Lys Asp Gly Ser Gly Lys 580 585 590 Leu Gly Leu Leu Glu Phe LysIle Leu Trp Lys Lys Leu Lys Lys Trp 595 600 605 Met Asp Ile Phe Arg GluCys Asp Gln Asp His Ser Gly Thr Leu Asn 610 615 620 Ser Tyr Glu Met ArgLeu Val Ile Glu Lys Ala Gly Ile Lys Leu Asn 625 630 635 640 Asn Lys ValMet Gln Val Leu Val Ala Arg Tyr Ala Asp Asp Asp Leu 645 650 655 Ile IleAsp Phe Asp Ser Phe Ile Ser Cys Phe Leu Arg Leu Lys Thr 660 665 670 MetPhe Met Ala Ala Asp Asp His Val Gly Ile Glu Ala Leu 675 680 685 9 2806DNA homo sapiens 9 caagcaccga gctagccacc agcatgctgt actccccagggccgagtctt ccggagtcag 60 cagagagcct ggatggatca caggaggata agcctcggggctcatgtgcg gagcccactt 120 ttactgatac gggaatggtg gctcacataa acaacagccggctcaaggcc aagggcgtgg 180 gccagcacga caacgcccag aactttggta accagagctttgaggagctg cgagcagcct 240 gtctaagaaa gggggagctc ttcgaggacc ccttattccctgctgaaccc agctcactgg 300 gcttcaagga cctgggcccc aactccaaaa atgtgcagaacatctcctgg cagcggccca 360 aggatatcat aaacaaccct ctattcatca tggatgggatttctccaaca gacatctgcc 420 aggggatcct cggggactgc tggctgctgg ctgccatcggctcccttacc acctgcccca 480 aactgctata ccgcgtggtg cccagaggac agagcttcaagaaaaactat gctggcatct 540 tccattttca gatttggcag tttggacagt gggtgaacgtggtggtagat gaccggctgc 600 ccacaaagaa tgacaagctg gtgtttgtgc actcaaccgaacgcagtgag ttctggagtg 660 ccctgctgga gaaggcgtat gccaagctga gtgggtcctatgaagcattg tcagggggca 720 gtaccatgga gggccttgag gacttcacag gaggcgtggcccagagcttc caactccaga 780 ggccccctca gaacctgctc aggctcctta ggaaggccgtggagcgatcc tccctcatgg 840 gttgctccat tgaagtcacc agtgatagtg aactggaatccatgactgac aagatgctgg 900 tgagagggca cgcttactct gtgactggcc ttcaggatgtccactacaga ggcaaaatgg 960 aaacactgat tcgggtccgg aatccctggg gccggattgagtggaatgga gcttggagtg 1020 acagtgccag ggagtgggaa gaggtggcct cagacatccagatgcagctg ctgcacaaga 1080 cggaggacgg ggagttctgg atgtcctacc aagatttcctgaacaacttc acgctcctgg 1140 agatctgcaa cctcacgcct gatacactct ctggggactacaagagctac tggcacacca 1200 ccttctacga gggcagctgg cgcagaggca gctccgcagggggctgcagg aaccaccctg 1260 gcacgttctg gaccaacccc cagtttaaga tctctcttcctgagggggat gacccagagg 1320 atgacgcaga gggcaatgtt gtggtctgca cctgcctggtggccctaatg cagaagaact 1380 ggcggcatgc acggcagcag ggagcccagc tgcagaccattggctttgtc ctctacgcgg 1440 tcccaaaaga gtttcagaac attcaggatg tccacttgaagaaggaattc ttcacgaagt 1500 atcaggacca cggcttctca gagatcttca ccaactcacgggaggtgagc agccaactcc 1560 ggctgcctcc gggggaatat atcattattc cctccacctttgagccacac agagatgctg 1620 acttcctgct tcgggtcttc accgagaagc acagcgagtcatgggaattg gatgaagtca 1680 actatgctga gcaactccaa gaggaaaagg tctctgaggatgacatggac caggacttcc 1740 tacatttgtt taagatagtg gcaggagagg gcaaggagataggggtgtat gagctccaga 1800 ggctgctcaa caggatggcc atcaaattca aaagcttcaagaccaagggc tttggcctgg 1860 atgcttgccg ctgcatgatc aacctcatgg ataaagatggctctggcaag ctggggcttc 1920 tagagttcaa gatcctgtgg aaaaaactca agaaatggatggacatcttc agagagtgtg 1980 accaggacca ttcaggcacc ttgaactcct atgagatgcgcctggttatt gagaaagcag 2040 gcatcaagct gaacaacaag gtaatgcagg tcctggtggccaggtatgca gatgatgacc 2100 tgatcataga ctttgacagc ttcatcagct gtttcctgaggctaaagacc atgttcacat 2160 tctttctaac catggacccc aagaatactg gccatatttgcttgagcctg gaacagtggc 2220 tgcagatgac catgtgggga tagaggcgct gtaggagcctggtcatctct accagcagca 2280 gcagcagcga ggttctagcc caggagggtg gggtgcttcttgtagccctc agctctccgg 2340 tctctgctga tgaaatgggc tccaggtggc agtgcccgggtcccaggtgc cgtgtttact 2400 gcagcagtgg gacctccgtg cccactcccc cagctcagaggctttctctt ttttccccaa 2460 cccggcttct gatggctggc tttcccccac catcgctctctcagagtata ttttactaaa 2520 gagtagttga tgcttcccca gggtccccct ggctggggaggccaagaata gggaagggac 2580 ttgtagcccg tttcttaccc tccatgcttg ctgtcctgctcacacctacc tgctgaccac 2640 ccatcctggc acagcctctg ttttcctccc catctgtggatactattcta ataaatagca 2700 catgccattg gcaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa 2760 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaa 2806

1.-3. (Cancelled)
 4. An isolated polypeptide comprising the amino acidsequence of SEQ ID NO:2.
 5. An antibody having immunospecificity for thepolypeptide sequence of SEQ ID NO:2.
 6. A process for identifying acompound useful for the treatment of low white blood cell count in apatient, comprising contacting a NHP preparation with said compound fora time sufficient to allow the compound to interact with or bind to theNHP preparation, and determining whether said compound inhibits theactivity of the NHP preparation.
 7. The process of claim 6, wherein saidNHP preparation comprises an isolated NIP protein, polypeptide orpeptide.
 8. The process of claim 6, wherein said NHP preparationcomprises a recombinant cell that expresses a NHP protein, polypeptide,or peptide.
 9. The process of claim 6, wherein said NHP preparationcomprises a transgenic nonhuman animal that expresses a human NHPprotein, polypeptide or peptide.
 10. The process of claim 6, whereinsaid NHP preparation comprises a human NHP protein, polypeptide, orpeptide.
 11. A process for treating low white blood cell count, in amammal in need of such treatment, comprising administering atherapeutically effective amount of a compound that inhibits NHPactivity to said mammal.
 12. The process of claim 11, wherein saidmammal is a human.
 13. A method for selectively inhibiting NHP activityin a human patient, comprising administering a compound that selectivityof a NHP protein, polypeptide or peptide in said patient, wherein theactivity of the compound does not result in significant toxic sideeffects in said patient.
 14. The method of claim 13, wherein theinhibition of the NHP protein, polypeptide or peptide increases whiteblood cell count in said patient.